Method of determining the identity and/or amount of an anti-il-31 antibody in a sample

ABSTRACT

The present invention provides a method of determining the identity and/or amount of an anti-IL-31 antibody in a sample. Such a method includes incubating a sample comprising an anti-IL-31 antibody with at least one mimotope selected from a feline IL-31 mimotope, a canine IL-31 mimotope, a horse IL-31 mimotope, and a human IL-31 mimotope; and determining the identity and/or quantity of the anti-IL-31 in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/356,505, filed Mar. 18, 2019, which claims the benefit of U.S.Provisional Application No. 62/643,921, filed Mar. 16, 2018, the entirecontents each of which are incorporated herein by reference in theirentirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (ZP000229B.xml; Size:246,770 bytes; and Date of Creation: Jul. 13, 2022) is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of peptide vaccines and theiruses in clinical and scientific procedures, including diagnosticprocedures. The peptide vaccines of the present invention are useful toimmunize and/or protect a mammal, such as a cat, dog, horse, or human,against an IL-31-mediated disorder.

BACKGROUND OF THE INVENTION

Atopic dermatitis has been defined by the American College of VeterinaryDermatology task force as “a genetically-predisposed inflammatory andpruritic allergic skin disease with characteristic clinical features”(Olivry, et al. Veterinary Immunology and Immunopathology 2001; 81:143-146). The task force also recognized that the disease in canines hasbeen associated with allergen-specific IgE (Olivry, et al. 2001 supra;Marsella & Olivry Clinics in Dermatology 2003; 21: 122-133). Severepruritus, along with secondary alopecia and erythema, are the mostnoticeable and concerning symptoms to pet owners.

The potential factors involved in allergic dermatitis are numerous andpoorly understood. Components in food may trigger atopic dermatitis(Picco, et al. Vet Dermatol. 2008; 19: 150-155), as well asenvironmental allergens such as fleas, dust mites, ragweed, plantextracts, etc. Genetic factors also play an important role. Althoughthere is no confirmed breed predilection, some mode of inheritance isthought to increase predisposition to atopic dermatitis (Sousa &Marsella Veterinary Immunology and Immunopathology 2001; 81: 153-157;Schwartzman, et al. Clin. Exp. Immunol. 1971; 9: 549-569).

The prevalence of atopic dermatitis is estimated to be 10% of the totalcanine population (Marsella & Olivry 2003 supra; Scott, et al. CanadianVeterinary Journal 2002; 43: 601-603; Hillier Veterinary Immunology andImmunopathology 2001; 81: 147-151). Globally, about 4.5 million dogs areaffected with this chronic and lifelong condition. Incidence appears tobe increasing. Canine breed and sex predilections have been suspected,but may vary greatly depending on geographical region (Hillier, 2001supra; Picco, et al. 2008 supra).

Feline allergic dermatitis is an inflammatory and pruritic skincondition thought to be caused by an abnormal response of the immunesystem to substances that do not induce a reaction in healthy cats. Themost consistent feature of feline allergic dermatitis is chronicrecurrent pruritus. Common clinical presentations of allergic dermatitisin cats include self-induced alopecia, miliary dermatitis, eosinophilicgranuloma complex lesions (including plaques, granulomas, and indolentulcer), and focused head and neck pruritus characterized byexcoriations, erosions, and/or ulcers. Breed and sex predilections havenot been demonstrated and young cats seem more prone to the disease(Hobi et al. Vet Dermatol 2011 22: 406-413; Ravens et al. Vet Dermatol2014; 25: 95-102; Buckely In Practice 2017; 39: 242-254).

Current treatments for cats diagnosed with allergic dermatitis depend onthe severity of the clinical signs, duration, and owner preferences andinclude allergen-specific immunotherapy and antipruritic drugs such asglucocorticoids and cyclosporines (Buckley, supra).

Immunotherapy treatment is effective for some patients but requiresfrequent injections, and clinical improvement may not be seen for 6-9months (Buckley, supra). Immunosuppressive drugs like glucocorticoidsand cyclosporines are generally effective however long term use oftenresults in undesirable adverse effects.

Atopic dermatitis in horses is recognized as a potential cause ofpruritus. The role of environmental allergens in equine atopicdermatitis is becoming better appreciated. The disease may be seasonalor non-seasonal, depending on the allergen(s) involved. Age, breed, andsex predilections have not been extensively reported. In preliminarywork at the School of Veterinary Medicine, University of California,Davis (SVM-UCD), the median age at onset was 6.5 years, Thoroughbredswere the most common breed, accounting for 25% of the horses, and males(usually geldings) were almost twice as prevalent as mares; however,these data are from only 24 horses, and have not yet been compared withthe hospital population at large. Pruritus, often directed against theface, distal legs, or trunk, is the most common clinical sign of equineatopic dermatitis. Alopecia, erythema, urticaria, and papules may all bepresent. Urticarial lesions may be quite severe, yet nonpruritic. Theremay be a familial predisposition for urticarial atopic dermatitis in thehorse. Horses may have a secondary pyoderma, typified by excess scaling,small epidermal collarettes, or encrusted papules (“miliarydermatitis”). Diagnosis of atopic dermatitis is based on clinical signsand the exclusion of other diagnoses, especially insect (Culicoides)hypersensitivity (White Clin Tech Equine Pract 2005; 4: 311-313; FadokVet Clin Equine 2013; 29 541-550). Currently, management of atopicdermatitis in horses is done both symptomatically, by suppressing theinflammation and the pruritus triggered by the allergic response, and byaddressing the specific cause (i.e., by identifying the responsibleallergens and by formulating an allergen-specific vaccine). Thesymptomatic approach is typically needed in the short term to make thepatient comfortable and minimize self-trauma. This approach relies onthe use of a combination of topical and systemic therapies includingantihistamines, essential fatty acids, pentoxifylline, andglucocorticoids. The primary approach to environmental allergy controlinvolves the identification of allergens that trigger thehypersensitivity reaction. It is commonly accepted by dermatologiststhat allergen-specific immunotherapy can be of help to atopic horses.However, as a general rule, most horses show improvement only after thefirst 6 months of immunotherapy (Marsella Vet Clin Equine 2013; 29:551-557). Also, long term use of immunosuppressive drugs in horses canresult in undesirable adverse effects.

Interleukin-31 (IL-31), a cytokine produced by T helper type 2 cells,has been shown to induce pruritus in humans, mice, and dogs (Bieber NEngl J Med 2008; 358: 1483-1494; Dillon et al. Nat Immunol 2004;5:752-60; U.S. Pat. No. 8,790,651 to Bammert et al.; Gonzalez et al. VetDermatl. 2013; 24(1): 48-53). IL-31 binds a co-receptor composed ofIL-31 receptor A (IL-31RA) and the oncostatin M receptor (OSMR) (Dillonet al. 2004 supra and Bilsborough et al. J Allergy Clin Immunol. 2006117(2):418-25). Receptor activation results in phosphorylation of STATthrough JAK receptor(s). Expression of the co-receptor has been shown inmacrophages, keratinocytes and in dorsal root ganglia.

Recently, it has been found that IL-31 is involved in dermatitis,pruritic skin lesions, allergy, and airway hypersensitivity. Cytopoint®,a canine anti-IL-31 monoclonal antibody produced by Zoetis Inc.,Parsippany, N.J., has been shown to reduce pruritus and skin lesions indogs with atopic dermatitis (Gonzalez et al. 2013 supra, Michels et al.Vet Dermatol. 2016; December; 27(6): 478-e129). It would be desirable toprovide for alternative approaches to prevent and treat IL-31-mediateddisorders in mammals. It would be especially desirable to providevaccines to reduce pruritus and skin lesions in dogs, cats, horses, andhumans with atopic dermatitis.

SUMMARY OF THE INVENTION

The present invention provides a method of determining the identityand/or amount of an anti-IL-31 antibody in a sample. Such a methodincludes incubating a sample comprising an anti-IL-31 antibody with atleast one mimotope selected from a feline IL-31 mimotope, a canine IL-31mimotope, a horse IL-31 mimotope, and a human IL-31 mimotope; anddetermining the identity and/or quantity of the anti-IL-31 in thesample. In one embodiment, the sample is from a vaccinated animal withan anti-IL-31 immune response. In another embodiment, the sample is froma mammal known to be or suspected of having a puritic and/or allergiccondition.

In one embodiment, the canine IL-31 mimotope employed in the method todetermine the identity and/or amount of an anti-IL-31 antibody in thesample is and/or comprises as part thereof the amino acid sequenceSVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187),NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192),APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) orvariants thereof that retain anti-IL-31 binding.

In another embodiment, the feline IL-31 mimotope employed in such amethod is and/or comprises as part thereof the amino acid sequenceSMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ IDNO: 193), APAHRLQPSDIRKIILELRPM SKG (SEQ ID NO: 197), IGLPES (SEQ ID NO:201) or variants thereof that retain anti-IL-31 binding.

In a further embodiment, the equine IL-31 mimotope employed in such amethod is and/or comprises as part thereof the amino acid sequenceSMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ IDNO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO:202) or variants thereof that retain anti-IL-31 binding.

In a still further, the human IL-31 mimotope employed in such a methodis and/or comprises as part thereof the amino acid sequence SVPTDTHECKRF(SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191),HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195),LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203) orvariants thereof that retain anti-IL-31 binding.

In one embodiment of the above-described diagnostic method, the mimotopeis a capture reagent bound to a solid surface. In a further embodiment,the sample is added to the mimotope capture reagent; and secondarydetection reagents are then added to quantify the amount of the antibodyin the sample.

In one embodiment of the above-described diagnostic method of theinvention, the mimotope binds to an anti-IL31 antibody orantigen-binding portion thereof that specifically binds to a region on amammalian IL-31 protein involved with interaction of the IL-31 proteinwith its co-receptor. In a further embodiment of the diagnostic methodof this invention, the binding of said antibody to said region isimpacted by mutations in a 15H05 epitope binding region selected fromthe group consisting of:

-   -   a) a region between about amino acid residues 124 and 135 of a        feline IL-31 sequence represented by SEQ ID NO: 157        (Feline_IL31_wildtype);    -   b) a region between about amino acid residues 124 and 135 of a        canine IL-31 sequence represented by SEQ ID NO: 155        (Canine_IL31); and    -   c) a region between about amino acid residues 118 and 129 of an        equine IL-31 sequence represented by SEQ ID NO: 165        (Equine_IL31).

In one specific embodiment of any of the diagnostic method of theinstant invention, the mimotope binds to an anti-IL-31 antibody orantigen-binding portion thereof comprising at least one of the followingcombinations of complementary determining region (CDR) sequences:

-   -   1) antibody 15H05: variable heavy (VH)-CDR1 of SYTIH (SEQ ID NO:        1), VH-CDR2 of NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 of        WGFKYDGEWSFDV (SEQ ID NO: 3), variable light (VL)-CDR1 of        RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 of KASNLHI (SEQ ID NO: 5),        and VL-CDR3 of LQSQTYPLT (SEQ ID NO: 6);    -   2) antibody ZIL1: variable heavy (VH)-CDR1 of SYGMS (SEQ ID NO:        13), VH-CDR2 of HINSGGSSTYYADAVKG (SEQ ID NO:14), VH-CDR3 of        VYTTLAAFWTDNFDY (SEQ ID NO: 15), variable light (VL)-CDR1 of        SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 of SDGNRPS (SEQ ID NO:        17, and VL-CDR3 of QSFDTTLDAYV (SEQ ID NO:18);    -   3) antibody ZIL8: VH-CDR1 of DYAMS (SEQ ID NO: 19), VH-CDR2 of        GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 of GFPGSFEH (SEQ ID        NO: 21), VL-CDR1 of TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 of        YNSDRPS (SEQ ID NO: 23), VL-CDR3 of SVYDRTFNAV (SEQ ID NO: 24);    -   4) antibody ZIL9: VH-CDR1 of SYDMT (SEQ ID NO: 25), VH-CDR2 of        DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 of LGVRDGLSV (SEQ ID        NO: 27), VL-CDR1 of SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 of        RDTERPS (SEQ ID NO: 29), VL-CDR3 of ESAVDTGTLV (SEQ ID NO: 30);    -   5) antibody ZIL11: VH-CDR1 of TYVMN (SEQ ID NO: 31), VH-CDR2 of        SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 of SMVGPFDY (SEQ ID        NO: 33), VL-CDR1 of SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 of        KDTERPS (SEQ ID NO: 35), VL-CDR3 of ESAVSSDTIV (SEQ ID NO: 36);    -   6) antibody ZIL69: VH-CDR1 of SYAMK (SEQ ID NO: 37), VH-CDR2 of        TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 of GNAESGCTGDHCPPY        (SEQ ID NO: 39), VL-CDR1 of SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2        of KDTERPS (SEQ ID NO: 41), VL-CDR3 of ESAVSSETNV (SEQ ID NO:        42);    -   7) antibody ZIL94: VH-CDR1 of TYFMS (SEQ ID NO: 43), VH-CDR2 of        LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 of FWRAFND (SEQ ID        NO: 45), VL-CDR1 of GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 of        DTGSRPS (SEQ ID NO: 47), VL-CDR3 of SLYTDSDILV (SEQ ID NO: 48);    -   8) antibody ZIL154: VH-CDR1 of DRGMS (SEQ ID NO: 49), VH-CDR2 of        YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 of WDGSSFDY (SEQ ID        NO: 51), VL-CDR1 of KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 of        KVSNRDP (SEQ ID NO: 53), VL-CDR3 of MQAIHFPLT (SEQ ID NO: 54);    -   9) antibody ZIL159: VH-CDR1 of SYVMT (SEQ ID NO: 55), VH-CDR2 of        GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 of GDIVATGTSY (SEQ ID        NO: 57), VL-CDR1 of SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 of        KDTERPS (SEQ ID NO: 59), VL-CDR3 of KSAVSIDVGV (SEQ ID NO: 60);    -   10) antibody ZIL171: VH-CDR1 of TYVMN (SEQ ID NO: 61), VH-CDR2        of SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 of SMVGPFDY (SEQ        ID NO: 63), VL-CDR1 of SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 of        KDTERPS (SEQ ID NO: 65), VL-CDR3 of ESAVSSDTIV (SEQ ID NO: 66);        or    -   11) a variant of 1) to 10) that differs from respective parent        antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154,        ZIL159, or ZIL171 by addition, deletion, and/or substitution of        one or more amino acid residues in at least one of VH or VL        CDR1, CDR2, or CDR3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an alignment showing amino acid sequence conservation betweenIL-31 from different species. In particular, a comparison between SEQ IDNO: 155 (canine IL-31), SEQ ID NO: 157 (feline IL-31), SEQ ID NO: 165(equine IL-31), and SEQ ID NO: 181 (human IL-31) is shown. In FIG. 1B,the percent amino acid sequence identity between canine, feline, horseand human IL-31 is also indicated.

FIG. 2 details the affinity with which candidate antibodies with CDRsderived from mouse origin bind feline and canine IL-31 using surfaceplasmon resonance (SPR) on a Biacore system (Biacore Life Sciences (GEHealthcare), Uppsala, Sweden).

FIG. 3 is a table showing potency (IC50 (μg/ml)) of candidate antibodieswith CDRs derived from mouse origin as measured by canine and felinecellular assays. In particular, the candidate antibodies were assessedfor their ability to inhibit IL-31-mediated STAT phosphorylation incanine DH-82 or feline FCWF4 macrophage-like cells.

FIG. 4 shows the results obtained for binding of candidate monoclonalantibodies with CDRs of dog origin to various proteins using both anindirect ELISA and Biacore methods. For the indirect ELISA, binding(ELISA OD) to wildtype feline IL-31 and a feline IL-31 15H05 mutantwhich had mutations in the monoclonal antibody 15H05 epitope region wasassessed. To confirm binding, biacore analysis was performed usingcanine, feline, equine, human, the feline 15H05 mutant, and feline 11E12mutant IL-31 proteins as surfaces and a single test concentration ofantibody. The feline IL-31 11E12 mutant had mutations in the monoclonalantibody 11E12 epitope region.

FIG. 5A shows an alignment of mouse antibody 11E12 VL sequence (SEQ IDNO: 73) comparing previously disclosed caninized 11E12 sequencesdesignated as Can_11E12_VL_cUn_1 (SEQ ID NO: 182) andCAN_11E12_VL_cUn_FW2 (SEQ ID NO: 184) to the felinized versionsdesignated as FEL_11E12_VH1 (SEQ ID NO: 111) and FEL_11E12_VL1_FW2 (SEQID NO: 117). Noted below the alignment in FIG. 5A are dots showing thepositons of relevant changes to Fel_11E12_VL1 that were necessary torestore affinity of this antibody to the IL-31 protein. FIG. 5B shows analignment of the mouse antibody 15H05 VL sequence designated herein asMU_15H05_VL (SEQ ID NO: 69) with the felinized 15H05 VL sequencesdesignated herein as FEl_15H05_VL1 (SEQ ID NO: 127) and FEl_15H05_VL_FW2(SEQ ID NO: 135). The dots below the alignment in FIG. 5B indicate thenecessary changes to the felinized 15H05 VL (Fel_15H05_VL1) that wererequired to not only restore, but improve, its affinity to canine andfeline IL-31 when compared to the mouse and chimeric forms of thisantibody.

FIG. 6A shows the alignment of wildtype feline IL-31 (SEQ ID NO: 157)with mutants 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161)highlighting the positions where the alanine substitutions occur. FIG.6B shows the feline IL-31 homology model highlighting the positions oftwo amino acids involved with binding of antibodies 11E12 (site 1) and15H05 (site 2). FIG. 6C is a graph showing the results obtained forbinding of monoclonal antibodies 11E12 and 15H05 to wild-type felineIL-31 and to mutant IL-31 proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQID NO: 161) when the wild-type and these mutants are used as the coatingantigens.

FIGS. 7A and 7B are of graphs showing competition binding assessments ofmAbs 15H05 and 11E12 using Biacore. FIG. 7A shows the competitionbinding data for mouse 15H05 and 11E12 antibodies to canine IL-31. FIG.7B shows the competition binding data for antibodies 15H05 and 11E12 ona feline IL-31 surface.

FIG. 8 is of a graph showing the results obtained for binding of theindividual receptor subunits of OSMR and IL-31Ra to wild-type felineIL-31 and to mutant IL-31 proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQID NO: 161) when the wild-type and these mutants are used as the coatingantigens.

FIG. 9 is of a graph showing the preliminary efficacy of mouse: feline11E12 chimera, mouse: feline 15H05 chimera, and felinized 11E12 (Feline11E12 1.1) in an IL-31 induced pruritus model in cats.

FIGS. 10A and 10B are of graphs showing the In vivo evaluation of theefficacy of a felinized15H05 anti IL-31 antibody termed ZTS-361 in a catpruritus challenge model. FIG. 10A shows the baseline pre-challengepruritic behavior for the T01 vehicle placebo and T02 antibody ZTS-361groups from day −7 through day 28 with day zero being the day ofantibody administration to group T02. FIG. 10B shows the efficacy ofantibody ZTS-361 demonstrating a significant reduction in pruritusobserved on days 7 (p<0.0001), 21 (p<0.0027), and 28 (p<0.0238)following IL-31 challenge when compared to vehicle placebo control.

FIG. 11A is of a graph showing the plasma levels of IL-31 in clientowned animals among dogs with atopic and allergic dermatitis compared tonormal laboratory FIG. 11B is of a graph showing the results of a recentstudy to determine serum IL-31 levels in cats with a presumptivediagnosis of allergic dermatitis (AD) from several different geographicregions in the USA. FIG. 11C is of a graph showing the pharmacokineticprofile of canine IL-31 in dogs following administration of asubcutaneous dose of 1.75 μg/kg canine IL-31.

FIG. 12 is of a table showing the results of a full replacement scan ofcanine IL-31 encompassing the amino acids outlined in FIG. 12. Eachposition depicted was individually replaced in the full-length canineIL-31 protein (SEQ ID NO: 155) with one of the other possible 19 aminoacids and binding of antibody 15H05 was assessed using an indirectELISA. For comparison, the corresponding region on feline (SEQ ID NO:157), equine (SEQ ID NO: 165), and human IL-31 (SEQ ID NO: 181) areshown.

FIG. 13A is of a table showing the sequences and chemical linkers ofvarious constrained peptides. Peptide ZTS-561 contains the amino acidsequence N-TEISVPADTFERKSFILT-C which corresponds to positions 121through 138 of SEQ ID NO: 155 with the substitution of Arginine (R) forCysteine (C) at position number 132. Peptide ZTS-562 contains the aminoacid sequence N-EISVPADTFERKSF-C which corresponds to positions 122through 135 of SEQ ID NO: 155 with the substitution of Arginine (R) forCysteine (C) at position number 132. Peptide ZTS-563 contains the aminoacid sequence N-AKVSMPADNFERKNFILT-C which corresponds to positions 121through 138 of SEQ ID NO: 157 with the substitution of Threonine (T) forAlanine (A) at position number 138. Peptide ZTS-564 contains the aminoacid sequence N-TEISVPADTFERKSFILT-C which corresponds to positions 121through 138 of SEQ ID NO: 155. Each of peptides ZTS-561, ZTS-562,ZTS-563, and ZTS-564 also includes N and C terminal Cysteines asdepicted to facilitate conjugation chemistry using the free thiolgroups. FIG. 13B shows the results of an affinity assessment for each ofpeptides ZTS-561, ZTS-562, ZTS-563, and ZTS-564 which had beenindependently conjugated to a carrier polypeptide (CRM-197). Foraffinity assessment, each peptide was independently immobilized to abiacore surface and the KD for the felinized anti IL-31 15H05 mAb(ZTS-927) was determined.

FIG. 14 depicts the study design for an immunogenicity study undertakento assess the ability of CRM-197-conjugated IL-31 mimotopes to generatean epitope-specific immune response driven towards the relevant regionon the IL-31 protein where antibody 15H05 and other anti-IL-31antibodies disclosed herein bind.

FIGS. 15A through 15E are of graphs showing serum titers generatedfollowing vaccination of dogs with IL-31 15H05 canine and felinemimotopes and full-length feline IL-31 protein organized by treatmentgroup showing the response at each day serum was taken. FIG. 15A depictsthe average canine antibody titers to full length feline IL-31 protein(SEQ ID NO: 159). FIG. 15B depicts the average canine antibody titers tothe full-length feline IL-31 15H05 mutant (SEQ ID NO: 163). FIG. 15Cdepicts the average canine antibody titers to full length canine IL-31(SEQ ID NO: 155). FIG. 15D depicts the average canine antibody titers tofull length equine IL-31 (SEQ ID NO: 165). FIG. 15E depicts the averagecanine antibody titers to full length human IL-31 (SEQ ID NO: 181).

FIG. 16A depicts the design for an immunogenicity study undertaken toassess the ability of CRM-197-conjugated full-length canine IL-31protein or mimotopes to elicit an immune response in laboratory beagledogs. Each mimotope described herein was designed to generate anepitope-specific immune response driven towards the relevant region onthe IL-31 protein where antibody 15H05 and other anti-IL-31 antibodiesdisclosed herein bind. The sequences and chemical linkers of variousmimotope peptides are shown as groups T02-T04. Peptide ZTS-420 containsthe amino acid sequence N-TEISVPADTFERKSFILT-C which corresponds topositions 121 through 138 of SEQ ID NO: 155 with the substitution ofArginine (R) for Cysteine (C) at position number 132. Peptide ZTS-421contains the amino acid sequence N-TNISVPTDTHECKRFILT-C whichcorresponds to positions 122 through 139 of SEQ ID NO: 181. PeptideZTS-766 contains the amino acid sequenceN-NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF-C which corresponds to positions 83through 115 of SEQ ID NO: 155. Each of peptides ZTS-420, ZTS-421, andZTS-766 also includes N and C terminal Cysteines as depicted tofacilitate conjugation chemistry using the free thiol groups. ZTS-766also contains an additional three amino acid spacer sequence (GSG) nextto the N terminal cysteine. FIG. 16B shows homologous sequences allowingcomparison of the canine BC helix mimotope (ZTS-766) to thecorresponding sequence from feline, equine, and human IL-31 and includesthe sequence reference number and amino acid positions for each.

FIGS. 17A and 17B are of graphs showing serum titers generated followingvaccination of dogs with IL-31 15H05 canine and human mimotopes, canineBC helix mimotope, and full-length feline IL-31 protein organized bytreatment group showing the response at each day serum was taken. Dogswere dosed on days 0, 28, and 56 indicated with arrows. FIG. 17A depictsthe average canine antibody titers to full length canine IL-31 protein(SEQ ID NO: 155). FIG. 17B depicts the average canine antibody titers tothe full length humanlL-31 (SEQ ID NO: 181) on days 0, 42, and 84 forgroup T03 only. Dogs in group T03 (human 15H05 mimotope) had no CRAR tocanine IL-31 (data not shown).

FIG. 18A depicts the design for an immunogenicity study undertaken toassess the ability of CRM-197-conjugated full-length feline IL-31protein or mimotopes to elicit an immune response in laboratory cats.All treatment groups were formulated with an adjuvant mixture includingthe glycolipid adjuvant Bay R1005(N-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamidehydroacetate)as well as CpG oligonucleotides. Each mimotope described herein wasdesigned to generate an epitope-specific immune response driven towardsthe relevant region on the IL-31 protein where antibody 15H05 and otheranti-IL-31 antibodies disclosed herein bind. The sequences and chemicallinkers of various mimotope peptides are shown as groups 102-105.Peptide ZTS-563 contains the amino acid sequence N-AKVSMPADNFERKNFILT-Cwhich corresponds to positions 121 through 138 of SEQ ID NO: 157 withthe substitution of Threonine (T) for Alanine (A) at position number138. Peptide ZTS-418 contains the amino acid sequenceN-TEVSMPTDNFERKRFILT-C which corresponds to positions 115 through 132 ofSEQ ID NO: 165. Peptide ZTS-423 contains the amino acid sequenceN-NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF-C which corresponds to positions 83through 115 of SEQ ID NO: 157. Peptide ZTS-422 contains the amino acidsequence N-AKVSMPADNFERKNFILT-C which corresponds to positions 121through 138 of SEQ ID NO: 157 with the substitution of Threonine (T) forAlanine (A) at position number 138. Each of peptides ZTS-563, ZTS-418,ZTS-423, and ZTS-422 also includes N and C terminal Cysteines asdepicted to facilitate conjugation chemistry using the free thiolgroups. ZTS-422 also contains an additional aminohexanoic acid linker(Ahx) between the two N terminal cysteines. ZTS-423 also contains anadditional three amino acid spacer sequence (GSG) next to the N terminalcysteine. FIG. 18B depicts the average feline antibody titers to thefull length feline IL-31 (SEQ ID NO: 157) for all treatment groupsexcept T03. Cats in group T03 (equine 15H05 mimotope) had no CRAR tofeline IL-31 (data not shown).

FIG. 19A is the minimum epitope amino acid sequence bound by anti-canineIL-31 antibody M14 according to WO 2018/156367 (Kindred Biosciences,Inc.) The comparison of multiple species, sequence reference IDs, andrelative amino acid positions are shown. FIG. 19B shows this minimumamino acid sequence on canine IL-31 highlighted in a black box. Thisfigure also shows the alignment of sequence in the surrounding region ofthe protein and the relative positions of the corresponding amino acidsin the sequence ID indicated.

FIG. 20 shows a fragment of the IL-31 protein from a loop formed by theconvergence of helix A with the trailing random coil sequence whichshares positional and structural attributes to the 15H05 loop.Comparison of the amino acid sequences from multiple species andreference to the sequence IDs and amino acid positions are shown.

FIG. 21A shows the amino acid sequences of three equine IL-31 mimotopepeptides representing different key epitope regions on the protein.Mimotope 15H05 contains the amino acid sequence N-TEVSMPTDNFERKRFILT-Cwhich corresponds to positions 115 through 132 of SEQ ID NO: 165.Mimotope BC helix contains the amino acid sequenceN-NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF-C which corresponds to positions 77through 109 of SEQ ID NO: 165. Mimotope A helix contains the amino acidsequence N-GPIYQLQPKEIQAIIVELQNLSKK-C which corresponds to positions 20through 43 of SEQ ID NO: 165. Mimotope 15H05 also includes N and Cterminal Cysteines as depicted to facilitate conjugation chemistry usingthe free thiol groups. All three mimotopes contain an additional threeamino acid spacer sequence (GSG) next to the N biotin group shown asbold and underlined in the sequences. The corresponding positions ofeach amino acid residue in SEQ ID NO: 165 are shown. FIG. 21B shows theresults from a binding assay using bio-layer interferometry. Themimotopes indicated were absorbed to streptavidin pins and used to probemultiple dilutions of mouse serum. The serum used was from micevaccinated with the equine IL-31 protein (SEQ ID NO: 165) or controlserum from mice vaccinated with an unrelated protein.

DEFINITIONS

Before describing the present invention in detail, several terms used inthe context of the present invention will be defined. In addition tothese terms, others are defined elsewhere in the specification, asnecessary. Unless otherwise expressly defined herein, terms of art usedin this specification will have their art-recognized meanings.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “an antibody” includes a pluralityof such antibodies. As another example, reference to “a mimotope”, “anIL-31 mimotope” and the like includes a plurality of such mimotopes.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers.

As used herein, the term “vaccine composition” includes at least oneantigen or immunogen in a pharmaceutically acceptable vehicle useful forinducing an immune response in a host. Vaccine compositions can beadministered in dosages, and by techniques well known to those skilledin the medical or veterinary arts, taking into consideration factorssuch as the age, sex, weight, species and condition of the recipientmammal, and the route of administration. The route of administration canbe percutaneous, via mucosal administration (e.g., oral, nasal, anal,vaginal) or via a parenteral route (intradermal, transdermal,intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccinecompositions can be administered alone, or can be co-administered orsequentially administered with other treatments or therapies. Forms ofadministration may include suspensions, syrups or elixirs, andpreparations for parenteral, subcutaneous, intradermal, intramuscular orintravenous administration (e.g., injectable administration) such assterile suspensions or emulsions. Vaccine compositions may beadministered as a spray, or mixed in food and/or water, or delivered inadmixture with a suitable carrier, diluent, or excipient such as sterilewater, physiological saline, glucose, or the like. The compositions cancontain auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, adjuvants, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standardpharmaceutical texts, such as “Remington's Pharmaceutical Sciences”(1990), may be consulted to prepare suitable preparations, without undueexperimentation.

The term “immune response” as used herein refers to a response elicitedin an animal or human. An immune response may refer to cellular immunity(CMI), humoral immunity, or may involve both. The present invention alsocontemplates a response limited to a part of the immune system. Usually,an “immunological response” includes, but is not limited to, one or moreof the following effects: the production or activation of antibodies, Bcells, helper T cells, suppressor T cells, and/or cytotoxic T cellsand/or yd T cells, directed specifically to an antigen or antigensincluded in the composition or vaccine of interest. Preferably, the hostwill display either a therapeutic or protective immunological response,such that resistance to the disease or disorder will be enhanced, and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an affected host, a quicker recovery time, and/or a loweredantigen (e.g., IL-31) titer in the affected host.

The term “protecting” as used herein means conferring a therapeuticimmunological response to a host mammal, such that resistance to adisease or disorder will be enhanced, and/or the clinical severity ofthe disease reduced in the host mammal.

As used herein, the term “immunogenicity” means capable of producing animmune response in a host mammal against an antigen or antigens. Thisimmune response forms the basis of the protective immunity elicited by avaccine against a specific antigen.

As used herein, immunizing, immunization, and the like is the processwhereby a mammal is made immune or resistant to a disease, typically bythe administration of a vaccine. Vaccines stimulate the mammal's ownimmune system to protect the mammal against subsequent disease.

An “adjuvant” as used herein means a composition comprised of one ormore substances that enhances the immune response to an antigen(s). Themechanism of how an adjuvant operates is not entirely known. Someadjuvants are believed to enhance the immune response by slowlyreleasing the antigen, while other adjuvants are strongly immunogenic intheir own right, and are believed to function synergistically.

Epitope, as used herein, refers to the antigenic determinant recognizedby the CDRs of the antibody. In other words, epitope refers to thatportion of any molecule capable of being recognized by, and bound by, anantibody. Unless indicated otherwise, the term “epitope” as used herein,refers to the region of IL-31 to which an anti-IL-31 agent is reactiveto.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of being recognizedby, and bound by, an antibody (the corresponding antibody binding regionmay be referred to as a paratope). In general, epitopes consist ofchemically active surface groupings of molecules, for example, aminoacids or sugar side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.Epitopes are the antigenic determinant on a protein that is recognizedby the immune system. The components of the immune system recognizingepitopes are antibodies, T-cells, and B-cells. T-cell epitopes aredisplayed on the surface of antigen-presenting cells (APCs) and aretypically 8-11 (MHC class I) or 15 plus (MHC class II) amino acids inlength. Recognition of the displayed MHC-peptide complex by T-cells iscritical to their activation. These mechanisms allow for the appropriaterecognition of self versus “non-self” proteins such as bacteria andviruses. Independent amino acid residues that are not necessarilycontiguous contribute to interactions with the APC binding cleft andsubsequent recognition by the T-Cell receptor (Janeway, Travers,Walport, Immunobiology: The Immune System in Health and Disease. 5thedition New York: Garland Science; 2001). Epitopes that are recognizedby soluble antibodies and cell surface associated B-cell receptors varygreatly in length and degree of continuity (Sivalingam and Shepherd,Immunol. 2012 July; 51(3-4):304-309 9). Again, even linear epitopes orepitopes found in a continuous stretch of protein sequence will oftenhave discontiguous amino acids that represent the key points of contactwith the antibody paratopes or B-cell receptor. Epitopes recognized byantibodies and B-cells can be conformational with amino acids comprisinga common area of contact on the protein in three-dimensional space andare dependent on tertiary and quaternary structural features of theprotein. These residues are often found in spatially distinct areas ofthe primary amino acid sequence.

A “mimotope” as used herein is a linear or constrained peptide whichmimics an antigen's epitope. A mimotope may have a primary amino acidsequence capable of eliciting a T-cell effector response and/or athree-dimensional structure necessary to bind B-cells resulting inmaturation of an acquired immunological response in an animal. Anantibody for a given epitope antigen will recognize a mimotope whichmimics that epitope. An IL-31 mimotope may alternatively be referred toherein as an IL-31 peptide mimotope. In some embodiments, a mimotope(linear or constrained) for use in the compositions and/or methods ofthe present invention is and/or comprises as part thereof a peptidewhich is from about 5 amino acid residues to about 40 amino acidresidues in length.

The term “specifically” in the context of antibody binding, refers tohigh avidity and/or high affinity binding of an antibody to a specificantigen, i.e., a polypeptide, or epitope. In many embodiments, thespecific antigen is an antigen (or a fragment or subfraction of anantigen) used to immunize the animal host from which theantibody-producing cells were isolated. Antibody specifically binding anantigen is stronger than binding of the same antibody to other antigens.Antibodies which bind specifically to a polypeptide may be capable ofbinding other polypeptides at a weak, yet detectable level (e.g., 10% orless of the binding shown to the polypeptide of interest). Such weakbinding, or background binding, is readily discernible from the specificantibody binding to a subject polypeptide, e.g. by use of appropriatecontrols. In general, specific antibodies bind to an antigen with abinding affinity with a K_(D) of 10⁻⁷ M or less, e.g., 10⁻⁸ M or less(e.g., 10⁻⁹ M or less, 10⁻¹⁰ or less, 10⁻¹¹ or less, 10⁻¹² or less, or10⁻¹³ or less, etc.).

As used herein, the term “antibody” refers to an intact immunoglobulinhaving two light and two heavy chains. Thus a single isolated antibodyor fragment may be a polyclonal antibody, a monoclonal antibody, asynthetic antibody, a recombinant antibody, a chimeric antibody, aheterochimeric antibody, a caninized antibody, a felinized antibody, afully canine antibody, a fully feline antibody, a fully equine antibody,or a fully human antibody. The term “antibody” preferably refers tomonoclonal antibodies and fragments thereof (e.g., including but notlimited to, antigen-binding portions of the antibody), and immunologicbinding equivalents thereof that can bind to the IL-31 protein andfragments or modified fragments thereof. Such fragments and modifiedfragments of IL-31 can include the IL-31 peptide mimotopes employed inthe various embodiments of this invention. For example, an antibody fora given epitope on IL-31 will recoginize an IL-31 peptide mimotope whichmimics that epitope. The term antibody is used both to refer to ahomogeneous molecular, or a mixture such as a serum product made up of aplurality of different molecular entities.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “antibody fragment” refers to less than an intact antibodystructure, including, without limitation, an isolated single antibodychain, an Fv construct, a Fab construct, an Fc construct, a light chainvariable or complementarity determining region (CDR) sequence, etc. Forexample, an antibody fragment can comprise the antigen-binding portionof the antibody.

The term “variable” region comprises framework and CDRs (otherwise knownas hypervariables) and refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise multiple FRs,largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the β-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat, et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), pages 647-669). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (Kabat, et al. (1991),above) and/or those residues from a “hypervariable loop” (Chothia andLesk J. Mol. Biol. 196:901-917 (1987). “Framework” or “FR” residues arethose variable domain residues other than the hypervariable regionresidues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.Presently there are five major classes of immunoglobulins: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 (asdefined by mouse and human designation). The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known in multiple species. The prevalence ofindividual isotypes and functional activities associated with theseconstant domains are species-specific and must be experimentallydefined.

“Monoclonal antibody” as defined herein is an antibody produced by asingle clone of cells (e.g., a single clone of hybridoma cells) andtherefore a single pure homogeneous type of antibody. All monoclonalantibodies produced from the same clone are identical and have the sameantigen specificity. The term “monoclonal” pertains to a single clone ofcells, a single cell, and the progeny of that cell.

“Fully canine antibody” as defined herein is a monoclonal antibodyproduced by a clone of cells (typically a CHO cell line) and therefore asingle pure homogeneous type of antibody. Antibodies identified fromsingle B cells of immunized mammals, such as dogs are created asrecombinant IgG proteins following identification of their variabledomain sequences. Grafting of these variable domains onto canineconstant domains (heavy chain and light chain kappa or lambda constant)results in the generation of recombinant fully canine antibodies. Allfully canine monoclonal antibodies produced from the same clone areidentical and have the same antigen specificity. The term “monoclonal”pertains to a single clone of cells, a single cell, and the progeny ofthat cell.

“Fully feline antibody” as defined herein is a monoclonal antibodyproduced by a clone of cells (typically a CHO cell line) and therefore asingle pure homogeneous type of antibody. Antibodies identified fromsingle B cells of immunized mammals, such as dogs are created asrecombinant IgG proteins following identification of their variabledomain sequences. Grafting of these variable domains onto felineconstant domains (heavy chain and light chain kappa or lambda constant)results in the generation of recombinant fully feline antibodies. Allfully feline monoclonal antibodies produced from the same clone areidentical and have the same antigen specificity. The term “monoclonal”pertains to a single clone of cells, a single cell, and the progeny ofthat cell.

“Fully equine antibody” as defined herein is a monoclonal antibodyproduced by a clone of cells (typically a CHO cell line) and therefore asingle pure homogeneous type of antibody. Antibodies identified fromsingle B cells of immunized mammals, such as dogs are created asrecombinant IgG proteins following identification of their variabledomain sequences. Grafting of these variable domains onto equineconstant domains (heavy chain and light chain kappa or lambda constant)results in the generation of recombinant fully equine antibodies. Allfully equine monoclonal antibodies produced from the same clone areidentical and have the same antigen specificity. The term “monoclonal”pertains to a single clone of cells, a single cell, and the progeny ofthat cell.

“Fully human antibody” as defined herein is a monoclonal antibodyproduced by a clone of cells (typically a CHO cell line) and therefore asingle pure homogeneous type of antibody. Antibodies identified fromsingle B cells of immunized mammals, such as dogs are created asrecombinant IgG proteins following identification of their variabledomain sequences. Grafting of these variable domains onto human constantdomains (heavy chain and light chain kappa or lambda constant) resultsin the generation of recombinant fully human antibodies. All fully humanmonoclonal antibodies produced from the same clone are identical andhave the same antigen specificity. The term “monoclonal” pertains to asingle clone of cells, a single cell, and the progeny of that cell.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species, while the remainder ofthe chain(s) is identical with or homologous to corresponding sequencesin antibodies derived from another species, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity.Typically, chimeric antibodies are antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to canine constant segments. In oneembodiment of a chimeric mouse:canine IgG, the antigen binding site isderived from mouse while the F_(c) portion is canine.

“Caninized” forms of non-canine (e.g., murine) antibodies aregenetically engineered antibodies that contain minimal sequence derivedfrom non-canine immunoglobulin. Caninized antibodies are canineimmunoglobulin sequences (recipient antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-canine species (donor antibody) such as mouse havingthe desired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the canine immunoglobulin sequencesare replaced by corresponding non-canine residues. Furthermore,caninized antibodies may include residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance. In general, the caninizedantibody will include substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-canine immunoglobulinsequence and all or substantially all of the FRs are those of a canineimmunoglobulin sequence. The caninized antibody optionally also willcomprise a complete, or at least a portion of an immunoglobulin constantregion (Fc), typically that of a canine immunoglobulin sequence. In oneembodiment of speciation or caninization of a mouse IgG, mouse CDRs aregrafted onto canine frameworks.

“Felinized” forms of non-feline (e.g., murine) antibodies aregenetically engineered antibodies that contain minimal sequence derivedfrom non-feline immunoglobulin. Felinized antibodies are felineimmunoglobulin sequences (recipient antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-feline species (donor antibody) such as mouse havingthe desired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the feline immunoglobulin sequencesare replaced by corresponding non-feline residues. Furthermore,felinized antibodies may include residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance. In general, the felinizedantibody will include substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-feline immunoglobulinsequence and all or substantially all of the FRs are those of a felineimmunoglobulin sequence. The felinized antibody optionally also willcomprise a complete, or at least a portion of an immunoglobulin constantregion (Fc), typically that of a feline immunoglobulin sequence.

“Equinized” forms of non-equine (e.g., murine) antibodies aregenetically engineered antibodies that contain minimal sequence derivedfrom non-equine immunoglobulin. Equinized antibodies are equineimmunoglobulin sequences (recipient antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-equine species (donor antibody) such as mouse havingthe desired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the equine immunoglobulin sequencesare replaced by corresponding non-equine residues. Furthermore,equinized antibodies may include residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance. In general, the equinizedantibody will include substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-equine immunoglobulinsequence and all or substantially all of the FRs are those of an equineimmunoglobulin sequence. The equinized antibody optionally also willcomprise a complete, or at least a portion of an immunoglobulin constantregion (Fc), typically that of an equine immunoglobulin sequence.

“Humanized” forms of non-human (e.g., murine) antibodies are geneticallyengineered antibodies that contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies are human immunoglobulinsequences (recipient antibody) in which hypervariable region residues ofthe recipient are replaced by hypervariable region residues from anon-human species (donor antibody) such as mouse having the desiredspecificity, affinity, and capacity. In some instances, framework region(FR) residues of the human immunoglobulin sequences are replaced bycorresponding non-human residues. Furthermore, humanized antibodies mayinclude residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin sequence and all orsubstantially all of the FRs are those of an human immunoglobulinsequence. The humanized antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of an human immunoglobulin sequence.

“Fully Canine” antibodies are genetically engineered antibodies thatcontain no sequence derived from non-canine immunoglobulin. Fully canineantibodies are canine immunoglobulin sequences (recipient antibody) inwhich hypervariable region residues are derived from a naturallyoccurring canine antibody (donor antibody) having the desiredspecificity, affinity, and capacity. In some instances, framework region(FR) residues of the canine immunoglobulin sequences are replaced bycorresponding non-canine residues. Furthermore, fully canine antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody, such as including, but not limited to changes in theCDRs to modify affinity. These modifications are made to further refineantibody performance. In general, the fully canine antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a canine immunoglobulin sequence and all orsubstantially all of the FRs are those of an canine immunoglobulinsequence. The fully canine antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of canine immunoglobulin sequence.

“Fully Feline” antibodies are genetically engineered antibodies thatcontain no sequence derived from non-feline immunoglobulin. Fully felineantibodies are feline immunoglobulin sequences (recipient antibody) inwhich hypervariable region residues are derived from a naturallyoccurring feline antibody (donor antibody) having the desiredspecificity, affinity, and capacity. In some instances, framework region(FR) residues of the feline immunoglobulin sequences are replaced bycorresponding non-feline residues. Furthermore, fully feline antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody, such as including, but not limited to changes in theCDRs to modify affinity. These modifications are made to further refineantibody performance. In general, the fully feline antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a feline immunoglobulin sequence and all orsubstantially all of the FRs are those of an feline immunoglobulinsequence. The fully feline antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of feline immunoglobulin sequence.

“Fully Equine” antibodies are genetically engineered antibodies thatcontain no sequence derived from non-equine immunoglobulin. Fully equineantibodies are equine immunoglobulin sequences (recipient antibody) inwhich hypervariable region residues are derived from a naturallyoccurring equine antibody (donor antibody) having the desiredspecificity, affinity, and capacity. In some instances, framework region(FR) residues of the equine immunoglobulin sequences are replaced bycorresponding non-equine residues. Furthermore, fully equine antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody, such as including, but not limited to changes in theCDRs to modify affinity. These modifications are made to further refineantibody performance. In general, the fully equine antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a equine immunoglobulin sequence and all orsubstantially all of the FRs are those of an equine immunoglobulinsequence. The fully equine antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of equine immunoglobulin sequence.

“Fully Human” antibodies are genetically engineered antibodies thatcontain no sequence derived from non-human immunoglobulin. Fully humanantibodies are human immunoglobulin sequences (recipient antibody) inwhich hypervariable region residues are derived from a naturallyoccurring human antibody (donor antibody) having the desiredspecificity, affinity, and capacity. In some instances, framework region(FR) residues of the human immunoglobulin sequences are replaced bycorresponding non-human residues. Furthermore, fully human antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody, such as including, but not limited to changes in theCDRs to modify affinity. These modifications are made to further refineantibody performance. In general, the fully human antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a human immunoglobulin sequence and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The fully human antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of human immunoglobulin sequence.

The term “heterochimeric” as defined herein, refers to an antibody inwhich one of the antibody chains (heavy or light) is, for example,caninized, felinized, equinized, or humanized while the other ischimeric. In one embodiment, a felinized variable heavy chain (where allof the CDRs are mouse and all FRs are feline) is paired with a chimericvariable light chain (where all of the CDRs are mouse and all FRs aremouse. In this embodiment, both the variable heavy and variable lightchains are fused to a feline constant region.

The term “variant” as used herein refers to a peptide, polypeptide or anucleic acid sequence encoding a peptide or polypeptide, that has one ormore conservative amino acid variations or other minor modificationssuch that the corresponding peptide or polypeptide has substantiallyequivalent function when compared to the wild-type peptide orpolypeptide. Ordinarily, variant peptide mimotopes for use in thepresent invention will have at least 30% identity to the parentmimotope, more preferably at least 50%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% sequenceidentity to the parent mimotope.

A “variant” anti-IL-31 antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” anti-IL-31 antibody aminoacid sequence by virtue of addition, deletion, and/or substitution ofone or more amino acid residue(s) in the parent antibody sequence andretains at least one desired activity of the parent anti-IL-31-antibody.Desired activities can include the ability to bind the antigenspecifically, the ability to reduce, inhibit or neutralize IL-31activity in an animal, and the ability to inhibit IL-31-mediated pSTATsignaling in a cell-based assay. In one embodiment, the variantcomprises one or more amino acid substitution(s) in one or morehypervariable and/or framework region(s) of the parent antibody. Forexample, the variant may comprise at least one, e.g. from about one toabout ten, and preferably from about two to about five, substitutions inone or more hypervariable and/or framework regions of the parentantibody. Ordinarily, the variant will have an amino acid sequencehaving at least 50% amino acid sequence identity with the parentantibody heavy or light chain variable domain sequences, more preferablyat least 65%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% sequence identity. Identity or homologywith respect to this sequence is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe parent antibody residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology. The variant retains theability to bind an IL-31 and preferably has desired activities which aresuperior to those of the parent antibody. For example, the variant mayhave a stronger binding affinity, enhanced ability to reduce, inhibit orneutralize IL-31 activity in an animal, and/or enhanced ability toinhibit IL-31-mediated pSTAT signaling in a cell-based assay.

A “variant” nucleic acid refers herein to a molecule which differs insequence from a “parent” nucleic acid. Polynucleotide sequencedivergence may result from mutational changes such as deletions,substitutions, or additions of one or more nucleotides. Each of thesechanges may occur alone or in combination, one or more times in a givensequence.

The “parent” antibody herein is one that is encoded by an amino acidsequence used for the preparation of the variant. In one embodiment, theparent antibody has a canine framework region and, if present, hascanine antibody constant region(s). For example, the parent antibody maybe a caninized or canine antibody. As another example, the parentantibody may be a felinized or feline antibody. As yet another example,the parent antibody may be an equinized or equine antibody. In anotherexample, the parent antibody may be a humanized or human antibody. In astill further example, the parent antibody is a murine monoclonalantibody.

The term “antigen binding region”, “antigen-binding portion”, and thelike as used throughout the specification and claims refers to thatportion of an antibody molecule which contains the amino acid residuesthat interact with an antigen and confer on the antibody its specificityand affinity for the antigen. The antibody binding region includes the“framework” amino acid residues necessary to maintain the properconformation of the antigen-binding residues. The antigen-bindingportion of an antibody according to the present invention mayalternatively be referred to herein as an IL-31-specific peptide orpolypeptide or as an anti-IL-31 peptide or polypeptide, for example.

The term “isolated” means that the material (e.g., antibody or nucleicacid) is separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses forthe material, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. With respect to nucleic acid, an isolatednucleic acid may include one that is separated from the 5′ to 3′sequences with which it is normally associated in the chromosome. Inpreferred embodiments, the material will be purified to greater than 95%by weight of the material, and most preferably more than 99% by weight.Isolated material includes the material in situ within recombinant cellssince at least one component of the material's natural environment willnot be present. Ordinarily, however, isolated material will be preparedby at least one purification step.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody,nucleic acid, or mimotope, for example. The label may itself bedetectable by itself (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition that is detectable.

The terms “nucleic acid”, “polynucleotide”, “nucleic acid molecule” andthe like may be used interchangeably herein and refer to a series ofnucleotide bases (also called “nucleotides”) in DNA and RNA. The nucleicacid may contain deoxyribonucleotides, ribonucleotides, and/or theiranalogs. The term “nucleic acid” includes, for example, single-strandedand double-stranded molecules. A nucleic acid can be, for example, agene or gene fragment, exons, introns, a DNA molecule (e.g., cDNA), anRNA molecule (e.g., mRNA), recombinant nucleic acids, plasmids, andother vectors, primers and probes. Both 5′ to 3′ (sense) and 3′ to 5′(antisense) polynucleotides are included.

A “subject” or “patient” refers to a mammal in need of treatment thatcan be affected by molecules of the invention. Mammals that can betreated in accordance with the invention include vertebrates, withmammals such as canine, feline, equine, and human mammals beingparticularly preferred examples.

A “therapeutically effective amount” (or “effective amount”) refers toan amount of an active ingredient, e.g., an agent according to theinvention, sufficient to effect beneficial or desired results whenadministered to a subject or patient. An effective amount can beadministered in one or more administrations, applications or dosages. Atherapeutically effective amount of a composition according to theinvention may be readily determined by one of ordinary skill in the art.In the context of this invention, a “therapeutically effective amount”is one that produces an objectively measured change in one or moreparameters associated with treatment of an IL-31 mediated disorder, suchas a pruritic condition or an allergic condition, or tumor progression,including clinical improvement in symptoms. Of course, thetherapeutically effective amount will vary depending upon the particularsubject and condition being treated, the weight and age of the subject,the severity of the disease condition, the particular compound chosen,the dosing regimen to be followed, timing of administration, the mannerof administration and the like, all of which can readily be determinedby one of ordinary skill in the art.

As used herein, the term “therapeutic” encompasses the full spectrum oftreatments for a disease or disorder. A “therapeutic” agent of theinvention may act in a manner that is prophylactic or preventive,including those that incorporate procedures designed to target animalsthat can be identified as being at risk (pharmacogenetics); or in amanner that is ameliorative or curative in nature; or may act to slowthe rate or extent of the progression of at least one symptom of adisease or disorder being treated.

“Treatment”, “treating”, and the like refers to both therapeutictreatment and prophylactic or preventative measures. Animals in need oftreatment include those already with the disorder as well as those inwhich the disorder is to be prevented. The term “treatment” or“treating” of a disease or disorder includes preventing or protectingagainst the disease or disorder (that is, causing the clinical symptomsnot to develop); inhibiting the disease or disorder (i.e., arresting orsuppressing the development of clinical symptoms; and/or relieving thedisease or disorder (i.e., causing the regression of clinical symptoms).As will be appreciated, it is not always possible to distinguish between“preventing” and “suppressing” a disease or disorder since the ultimateinductive event or events may be unknown or latent. Accordingly, theterm “prophylaxis” will be understood to constitute a type of“treatment” that encompasses both “preventing” and “suppressing.” Theterm “treatment” thus includes “prophylaxis”.

The term “allergic condition” is defined herein as a disorder or diseasecaused by an interaction between the immune system and a substanceforeign to the body. This foreign substance is termed “an allergen”.Common allergens include aeroallergens, such as pollens, dust, molds,dust mite proteins, injected saliva from insect bites, etc. Examples ofallergic conditions include, but are not limited to, the following:allergic dermatitis, summer eczema, urticaria, heaves, inflammatoryairway disease, recurrent airway obstruction, airwayhyper-responsiveness, chronic obstructive pulmonary disease, andinflammatory processes resulting from autoimmunity, such as Irritablebowel syndrome (IBS).

The term “pruritic condition” is defined herein as a disease or disordercharacterized by an intense itching sensation that produces the urge torub or scratch the skin to obtain relief. Examples of pruriticconditions include, but are not limited to the following: atopicdermatitis, allergic dermatitis, eczema, psoriasis, scleroderma, andpruritus.

As used herein, the terms “cell”, “cell line”, and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells,yeast cells, mammalian cells, and insect cells) whether located in vitroor in vivo. For example, host cells may be located in a transgenicanimal. Host cell can be used as a recipient for vectors and may includeany transformable organism that is capable of replicating a vectorand/or expressing a heterologous nucleic acid encoded by a vector.

A “composition” is intended to mean a combination of active agent andanother compound or composition which can be inert (e.g., a label), oractive, such as an adjuvant.

As used herein, the terms “pharmaceutically acceptable carrier” and“pharmaceutically acceptable vehicle” are interchangeable, and refer toa fluid vehicle for containing vaccine antigens that can be injectedinto a host without adverse effects. Pharmaceutically acceptablecarriers suitable for use in the invention are well known to those ofskill in the art. Such carriers include, without limitation, water,saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions,emulsions or suspensions. Other conventionally employed diluents,adjuvants and excipients, may be added in accordance with conventionaltechniques. Such carriers can include ethanol, polyols, and suitablemixtures thereof, vegetable oils, and injectable organic esters. Buffersand pH adjusting agents may also be employed. Buffers include, withoutlimitation, salts prepared from an organic acid or base. Representativebuffers include, without limitation, organic acid salts, such as saltsof citric acid, e.g., citrates, ascorbic acid, gluconic acid,histidine-HCl, carbonic acid, tartaric acid, succinic acid, acetic acid,or phthalic acid, Tris, trimethanmine hydrochloride, or phosphatebuffers. Parenteral carriers can include sodium chloride solution,Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride,lactated Ringer's or fixed oils. Intravenous carriers can include fluidand nutrient replenishers, electrolyte replenishers, such as those basedon Ringer's dextrose and the like. Preservatives and other additivessuch as, for example, antimicrobials, antioxidants, chelating agents(e.g., EDTA), inert gases and the like may also be provided in thepharmaceutical carriers. The present invention is not limited by theselection of the carrier. The preparation of these pharmaceuticallyacceptable compositions, from the above-described components, havingappropriate pH isotonicity, stability and other conventionalcharacteristics is within the skill of the art. See, e.g., texts such asRemington: The Science and Practice of Pharmacy, 20th ed, LippincottWilliams & Wilkins, publ., 2000; and The Handbook of PharmaceuticalExcipients, 4.sup.th edit., eds. R. C. Rowe et al, APhA Publications,2003.

The term “conservative amino acid substitution” indicates any amino acidsubstitution for a given amino acid residue, where the substituteresidue is so chemically similar to that of the given residue that nosubstantial decrease in polypeptide function (e.g., enzymatic activity)results. Conservative amino acid substitutions are commonly known in theart and examples thereof are described, e.g., in U.S. Pat. Nos.6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferredembodiment, a conservative amino acid substitution will be any one thatoccurs within one of the following six groups

-   -   1. Small aliphatic, substantially non-polar residues: Ala, Gly,        Pro, Ser, and Thr;    -   2. Large aliphatic, non-polar residues: Ile, Leu, and Val; Met;    -   3. Polar, negatively charged residues and their amides: Asp and        Glu;    -   4. Amides of polar, negatively charged residues: Asn and Gln;        His;    -   5. Polar, positively charged residues: Arg and Lys; His; and    -   6. Large aromatic residues: Trp and Tyr; Phe.    -   In a preferred embodiment, a conservative amino acid        substitution will be any one of the following, which are listed        as Native Residue (Conservative Substitutions) pairs: Ala (Ser);        Arg (Lys); Asn (Gln; His); Asp (Glu); Gln (Asn); Glu (Asp); Gly        (Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg;        Gln; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser (Thr); Thr        (Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (Ile; Leu).

Just as a polypeptide may contain conservative amino acidsubstitution(s), a polynucleotide hereof may contain conservative codonsubstitution(s). A codon substitution is considered conservative if,when expressed, it produces a conservative amino acid substitution, asdescribed above. Degenerate codon substitution, which results in noamino acid substitution, is also useful in polynucleotides according tothe present invention. Thus, e.g., a polynucleotide encoding a selectedpolypeptide useful in an embodiment of the present invention may bemutated by degenerate codon substitution in order to approximate thecodon usage frequency exhibited by an expression host cell to betransformed therewith, or to otherwise improve the expression thereof.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Unless otherwise defined, scientific and technical terms used inconnection with the vaccine compositions and antibodies described hereinshall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well-known and commonly used in the art.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transfection (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification, See e.g., Sambrook et al. MOLECULAR CLONING: LAB. MANUAL(3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y., 2001)and Ausubel et al. Current Protocols in Molecular Biology (New York:Greene Publishing Association/Wiley Interscience), 1993. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application.

Compositions

The present invention provides for IL-31 mimotopes (peptides) andvariants thereof and their uses in clinical and scientific procedures,including diagnostic procedures. As used herein, such an IL-31 mimotopeis a linear or constrained peptide which mimics an antigen's epitope. Ananti-IL-31 antibody for a given IL-31 epitope antigen will recognize anIL-31 mimotope which mimics that epitope.

IL-31 mimotopes (peptides) are employed in vaccine compositionsaccording to the present invention. Such vaccine compositions are usefulfor protecting a mammal against an IL-31 mediated disorder, such as apruritic or allergic condition. In some embodiments, the IL-31-mediatedpruritic or allergic condition is a pruritic condition selected fromatopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. Inother embodiments, the IL-31-mediated pruritic or allergic condition isan allergic condition selected from allergic dermatitis, summer eczema,urticaria, heaves, inflammatory airway disease, recurrent airwayobstruction, airway hyper-responsiveness, chronic obstruction pulmonarydisease, and inflammatory processes resulting from autoimmunity. Inother embodiments, the IL-31 mediated disorder is tumor progression. Insome embodiments, the IL-31 mediated disorder is eosinophilic disease ormastocytomas.

In one embodiment, a vaccine composition according to the presentinvention includes the combination of a carrier polypeptide and at leastone mimotope selected from a feline IL-31 mimotope, a canine IL-31mimotope, a horse IL-31 mimotope, and a human IL-31 mimotope; and anadjuvant. In some embodiments, the vaccine compositions of thisinvention can include more than one IL-31 mimotope from a given species,or even a combination of IL-31 mimotopes from different species. In someembodiments, a mimotope (linear or constrained) for use in thecompositions and/or methods of the present invention is and/or comprisesas part thereof a peptide which is from about 5 amino acid residues toabout 40 amino acid residues in length.

In one embodiment, the at least one mimotope employed in thecompositions and methods of the instant invention is selected from anIL-31 15H05 mimotope, an IL-31 helix BC region mimotope, an IL-31 helixA region mimotope, an IL-31 AB loop region mimotope, or any combinationthereof.

In one embodiment, the at least one mimotope for use in the compositionsand methods of the instant invention generates antibodies that areneutralizing the bioactivity of IL-31.

In another embodiment, the vaccine compositions of this invention arecapable of eliciting a focused immune response to generate antibodies inthe mammal directed to at least one neutralizing epitope on IL-31, butnot against non-neutralizing epitopes on IL-31.

In one embodiment, the vaccine composition includes a canine IL-31mimotope which is and/or includes as part thereof the amino acidsequence SVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187),NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192),APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) orvariants thereof that retain anti-IL-31 binding.

In another embodiment, the vaccine composition includes a feline IL-31mimotope which is and/or includes as part thereof the amino acidsequence SMPADNFERKNF (SEQ ID NO: 188),NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193),APAHRLQPSDIRKIILELRPMSKG (SEQ ID NO: 197), IGLPES (SEQ ID NO: 201) orvariants thereof that retain anti-IL-31 binding.

In yet another embodiment, the vaccine composition includes an equineIL-31 mimotope which is and/or includes as part thereof the amino acidsequence SMPTDNFERKRF (SEQ ID NO: 189),NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194),GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO: 202) orvariants thereof that retain anti-IL-31 binding.

In a still further embodiment, the vaccine composition includes a humanIL-31 mimotope which is and/or includes as part thereof the amino acidsequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191),HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195),LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203) orvariants thereof that retain anti-IL-31 binding.

In one embodiment, the mimotope employed in the vaccine compositionsaccording to the present invention is a constrained mimotope. In oneembodiment, such a constrained mimotope is a chemically-linked cyclicpeptide.

Linear IL-31 mimotopes can be chemically synthesized or recombinantlyproduced. Constrained IL-31 mimotopes such as a chemically-linked cyclicpeptide can be chemically synthesized or can be made using a combinationof chemical synthesis and recombinant technology. In some embodiments,the IL-31 mimetope employed in the vaccine composition is chemicallyconjugated to a carrier polypeptide. In other embodiments, the carrierpolypeptide and the mimotope are part of a recombinant fusion protein.

The carrier polypeptide which is combined with the IL-31 mimotope can beor can include as part thereof a bacterial toxoid or a derivativethereof, keyhole limpet hemocyanin (KLH), or a virus-like particle. Byway of non-limiting examples, the bacterial toxoid or derivative can bea tetanus toxoid, a diphtheria toxoid, a tetanus toxoid, the outermembrane protein complex from group B N. meningitidis, Pseudomonasexotoxin, or the nontoxic mutant of diphtheria toxin (CRM197). By way ofother non-limiting examples, the virus-like particle can be HBsAg,HBcAg, E. coli bacteriophage Qbeta, Norwalk virus, canine distempervirus (CDV), or influenza HA. In one preferred embodiment, the IL-31mimotope is in a combination with a carrier polypeptide which includesor consists of CRM197.

The vaccine compositions according to the present invention include atleast one adjuvant or adjuvant formulation, as will be described infurther detail below.

Vaccines of the present invention can be formulated following acceptedconvention to include pharmaceutically acceptable carriers for animals,including humans (if applicable), such as standard buffers, stabilizers,diluents, preservatives, and/or solubilizers, and can also be formulatedto facilitate sustained release. Diluents include water, saline,dextrose, ethanol, glycerol, and the like. Additives for isotonicityinclude sodium chloride, dextrose, mannitol, sorbitol, and lactose,among others. Stabilizers include albumin, among others. Other suitablevaccine vehicles and additives, including those that are particularlyuseful in formulating modified live vaccines, are known or will beapparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

Vaccines of the present invention can further comprise one or moreadditional immunomodulatory components such as, e.g., an adjuvant orcytokine, among others. Types of suitable adjuvants for use in thecompositions of the present invention include the following: anoil-in-water adjuvant, a polymer and water adjuvant, a water-in-oiladjuvant, an aluminum hydroxide adjuvant, a vitamin E adjuvant andcombinations thereof. Some specific examples of adjuvants include, butare not limited to, complete Freund's adjuvant, incomplete Freund'sadjuvant, Corynebacterium parvum, Bacillus Calmette Guerin, aluminumhydroxide gel, glucan, dextran sulfate, iron oxide, sodium alginate,Bacto-Adjuvant, certain synthetic polymers such as poly amino acids andco-polymers of amino acids, Block copolymer (CytRx, Atlanta, Ga.), QS-21(Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, EmeryvilleCalif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction,monophosphoryl lipid A, and Avridine lipid-amine adjuvant(N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), “REGRESSIN”(Vetrepharm, Athens, Ga.), paraffin oil, RIBI adjuvant system (RibiInc., Hamilton, Mont.), muramyl dipeptide and the like.

Non-limiting examples of oil-in-water emulsions useful in the vaccine ofthe invention include modified SEAM62 and SEAM 1/2 formulations.Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene(Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v)TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/mlQuil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v)SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol,100 μg/ml Quil A, and 50 μg/ml cholesterol.

Another example of an adjuvant useful in the compositions of theinvention is SP-oil. As used in the specification and claims, the term“SP oil” designates an oil emulsion comprising apolyoxyethylene-polyoxypropylene block copolymer, squalane,polyoxyethylene sorbitan monooleate and a buffered salt solution.Polyoxyethylene-polyoxypropylene block copolymers are surfactants thataid in suspending solid and liquid components. These surfactants arecommercially available as polymers under the trade name Pluronic®. Thepreferred surfactant is poloxamer 401 which is commercially availableunder the trade name Pluronic® L-121. In general, the SP oil emulsion isan immunostimulating adjuvant mixture which will comprise about 1 to 3%vol/vol of block copolymer, about 2 to 6% vol/vol of squalane, moreparticularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol ofpolyoxyethylene sorbitan monooleate, with the remainder being a bufferedsalt solution.

“Immunomodulators” that can be included in the vaccine include, e.g.,immunostimulatory oligonucleotides, one or more interleukins,interferons, or other known cytokines. In one embodiment, the adjuvantmay be a cyclodextrin derivative or a polyanionic polymer, such as thosedescribed in U.S. Pat. Nos. 6,165,995 and 6,610,310, respectively.

In one embodiment, the adjuvant is a formulation comprising a saponin, asterol, a quaternary ammonium compound, and a polymer. In a specificembodiment, the saponin is Quil A or a purified fraction thereof, thesterol is cholesterol, the quaternary ammonium compound is dimethyldioctadecyl ammonium bromide (DDA), and the polymer is polyacrylic acid.

In another embodiment, the adjuvant comprises the combination of one ormore isolated immunostimulatory oligonucleotides, a sterol, and asaponin. In a specific embodiment, the one or more isolatedimmunostimulatory oligonucleotides comprises CpG, the sterol ischolesterol, and the saponin is Quil A or a purified fraction thereof.As used herein, the ZA-01 adjuvant referred to in the example sectionincludes Quil A (saponin), cholesterol, CpG, and diluent.

In another embodiment, a useful adjuvant to be employed in thecompositions of this invention includes CpG-containing immunostimulatoryoligonucleotides. CpG-containing oligonucleotides are described forexample in U.S. Pat. No. 8,580,280. In one specific embodiment, anadjuvant for use in the present invention is a mixture including atleast one glycolipid adjuvant and CpG-containing oligonucleotides. Aspecific example of a useful adjuvant is a mixture that includes theglycolipid adjuvant Bay R1005(N-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamidehydroacetate)as well as CpG oligonucleotides.

In one embodiment, the adjuvant or adjuvant mixture is added in anamount of about 100 μg to about 10 mg per dose. In another embodiment,the adjuvant/adjuvant mixture is added in an amount of about 200 μg toabout 5 mg per dose. In yet another embodiment, the adjuvant/adjuvantmixture is added in an amount of about 300 μg to about 1 mg/dose.

With the advent of methods of molecular biology and recombinanttechnology, it is possible to produce the aforementioned peptides andpolypeptides by recombinant means and thereby generate gene sequencesthat code for specific amino acid sequences found in the peptide orpolypeptide structure. In one embodiment, the peptide is the IL-31mimotope or is at least part of the IL-31 mimotope. In anotherembodiment, the polypeptide is the carrier polypeptide which is presentin combination with the IL-31 mimotope. In a still further embodiment,the polypeptide is an antibody, such as that to which the IL-31 mimotopeemployed in the vaccine composition or diagnostic methods of thisinvention binds. Such antibodies can be produced by either cloning thegene sequences encoding the polypeptide chains of said antibodies or bydirect synthesis of said polypeptide chains, with assembly of thesynthesized chains to form active tetrameric (H₂L₂) structures withaffinity for specific epitopes and antigenic determinants. This haspermitted the ready production of antibodies having sequencescharacteristic of neutralizing antibodies from different species andsources.

Regardless of the source of the antibodies, or how they arerecombinantly-constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, large cell cultures of laboratory orcommercial size, using transgenic plants, or by direct chemicalsynthesis employing no living organisms at any stage of the process, allantibodies have a similar overall 3 dimensional structure. Thisstructure is often given as H₂L₂ and refers to the fact that antibodiescommonly comprise two light (L) amino acid chains and 2 heavy (H) aminoacid chains. Both chains have regions capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or “V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity. The variable regions of either H or Lchains contain the amino acid sequences capable of specifically bindingto antigenic targets.

The “antigen binding region”, or “antigen-binding portion” of anantibody refers to that portion of an antibody molecule which containsthe amino acid residues that interact with an antigen and confer on theantibody its specificity and affinity for the antigen. The antibodybinding region includes the “framework” amino acid residues necessary tomaintain the proper conformation of the antigen-binding residues. Theantigen-binding portion of an antibody referred to in the specificationand claims may be referred to herein as an IL-31-specific peptide orpolypeptide or as an anti-IL-31 peptide or polypeptide, for example.

Within the variable regions of the H or L chains that provide for theantigen binding regions are smaller sequences dubbed “hypervariable”because of their extreme variability between antibodies of differingspecificity. Such hypervariable regions are also referred to as“complementarity determining regions” or “CDR” regions. These CDRregions account for the basic specificity of the antibody for aparticular antigenic determinant structure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have three CDR regions, eachnon-contiguous with the others.

In all mammalian species, antibody peptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

Regarding the antigenic determinate recognized by the CDR regions of theantibody, this is also referred to as the “epitope.” In other words,epitope refers to that portion of any molecule capable of beingrecognized by, and bound by, an antibody (the corresponding antibodybinding region may be referred to as a paratope).

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce an antibody capable of binding to an epitope of that antigen.An antigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibodies referred to herein are meant to include both intactimmunoglobulin molecules as well as portions, fragments, peptides andderivatives thereof such as, for example, Fab, Fab′, F(ab′)₂, Fv, Fse,CDR regions, paratopes, or any portion (e.g., a polypeptide) or peptidesequence of the antibody that is capable of binding an antigen orepitope. An antibody is said to be “capable of binding” a molecule if itis capable of specifically reacting with the molecule to thereby bindthe molecule to the antibody.

The antibodies referred to herein also include chimeric antibodies,heterochimeric antibodies, caninized antibodies, felinized antibodies,equinized antibodies, humanized antibodies, fully canine antibodies,fully feline antibodies, fully equine antibodies, fully humanantibodies, as well as fragments, portions, regions, peptides orderivatives thereof, provided by any known technique, such as, but notlimited to, enzymatic cleavage, peptide synthesis, or recombinanttechniques. Such antibodies referred to herein are capable ofspecifically binding at least one of canine IL-31, feline IL-31, equineIL-31, or human IL-31. Antibody fragments or portions may lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody.Examples of antibody fragments may be produced from intact antibodiesusing methods well known in the art, for example by proteolytic cleavagewith enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). See, e.g., Wahl et al., 24 J. Nucl. Med.316-25 (1983). Portions of antibodies may be made by any of the abovemethods, or may be made by expressing a portion of the recombinantmolecule. For example, the CDR region(s) of a recombinant antibody maybe isolated and subcloned into the appropriate expression vector. See,e.g., U.S. Pat. No. 6,680,053.

Clones 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 andZIL171 Nucleotide and Amino Acid Sequences

In some embodiments, the present invention provides for IL-31 mimotopeswhich bind to novel monoclonal antibodies that specifically bind to atleast one of canine IL-31, feline IL-31, or equine IL-31. Suchmonoclonal antibodies may be employed in the diagnostic methods of thisinvention together with the IL-31 mimotope. In one embodiment, amonoclonal antibody referred to in the specification and claims binds tocanine IL-31, feline IL-31, or equine IL-31 and prevents its binding to,and activation of, its co-receptor complex comprising IL-31 receptor A(IL-31Ra) and Oncostatin-M-specific receptor (OsmR or IL-31Rb). Examplesof such monoclonal antibodies are identified herein as “15H05”, “ZIL1”,“ZIL8”, “ZIL9”, “ZIL11”, “ZIL69”, “ZIL94”, “ZIL154”, “ZIL159” and“ZIL171”, which refers to the number assigned to its clone. Herein,“15H05”, “ZIL1”, “ZIL8”, “ZIL9”, “ZIL11”, “ZIL69”, “ZIL94”, “ZIL154”,“ZIL159” and “ZIL171” also refers to the portion of the monoclonalantibody, the paratope or CDRs, that bind specifically with an IL-31epitope identified as 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94,ZIL154, ZIL159 and ZIL171 because of its ability to bind the 15H05,ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 and ZIL171antibodies, respectively. The several recombinant, chimeric,heterochimeric, caninized, felinized, equinized, fully canine, fullyfeline, and/or fully equine forms of 15H05, ZIL1, ZIL8, ZIL9, ZIL11,ZIL69, ZIL94, ZIL154, ZIL159 and ZIL171 described herein may be referredto by the same name.

In one embodiment, a vaccine composition according to the presentinvention includes a mimotope which binds to an anti-IL31 antibody orantigen-binding portion thereof that specifically binds to a region on amammalian IL-31 protein involved with interaction of the IL-31 proteinwith its co-receptor. In one embodiment, the binding of said antibody tosaid region is impacted by mutations in a 15H05 epitope binding regionselected from: a) a region between about amino acid residues 124 and 135of a feline IL-31 sequence represented by SEQ ID NO: 157(Feline_IL31_wildtype); b) a region between about amino acid residues124 and 135 of a canine IL-31 sequence represented by SEQ ID NO: 155(Canine_IL31); and c) a region between about amino acid residues 118 and129 of an equine IL-31 sequence represented by SEQ ID NO: 165(Equine_IL31). In one embodiment, the mimotope for use in thecompositions of the present invention binds to an anti-IL-31 antibody orantigen-binding portion thereof that specifically binds to theaforementioned 15H05 epitope region.

In one particular embodiment of a vaccine composition according to thisinvention, the mimotope binds to an anti-IL-31 antibody orantigen-binding portion thereof which includes at least one of thefollowing combinations of complementary determining region (CDR)sequences:

1) antibody 15H05: variable heavy (VH)-CDR1 of SYTIH (SEQ ID NO: 1),VH-CDR2 of NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 of WGFKYDGEWSFDV(SEQ ID NO: 3), variable light (VL)-CDR1 of RASQGISIWLS (SEQ ID NO: 4),VL-CDR2 of KASNLHI (SEQ ID NO: 5), and VL-CDR3 of LQSQTYPLT (SEQ ID NO:6);

2) antibody ZIL1: variable heavy (VH)-CDR1 of SYGMS (SEQ ID NO: 13),VH-CDR2 of HINSGGSSTYYADAVKG (SEQ ID NO:14), VH-CDR3 of VYTTLAAFWTDNFDY(SEQ ID NO: 15), variable light (VL)-CDR1 of SGSTNNIGILAAT (SEQ ID NO:16), VL-CDR2 of SDGNRPS (SEQ ID NO: 17, and VL-CDR3 of QSFDTTLDAYV (SEQID NO:18);

3) antibody ZIL8: VH-CDR1 of DYAMS (SEQ ID NO: 19), VH-CDR2 ofGIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 of GFPGSFEH (SEQ ID NO: 21),VL-CDR1 of TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 of YNSDRPS (SEQ ID NO:23), VL-CDR3 of SVYDRTFNAV (SEQ ID NO: 24);

4) antibody ZIL9: VH-CDR1 of SYDMT (SEQ ID NO: 25), VH-CDR2 ofDVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 of LGVRDGLSV (SEQ ID NO: 27),VL-CDR1 of SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 of RDTERPS (SEQ ID NO:29), VL-CDR3 of ESAVDTGTLV (SEQ ID NO: 30);

5) antibody ZIL11: VH-CDR1 of TYVMN (SEQ ID NO: 31), VH-CDR2 ofSINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 of SMVGPFDY (SEQ ID NO: 33),VL-CDR1 of SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 of KDTERPS (SEQ ID NO:35), VL-CDR3 of ESAVSSDTIV (SEQ ID NO: 36);

6) antibody ZIL69: VH-CDR1 of SYAMK (SEQ ID NO: 37), VH-CDR2 ofTINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 of GNAESGCTGDHCPPY (SEQ IDNO: 39), VL-CDR1 of SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 of KDTERPS (SEQID NO: 41), VL-CDR3 of ESAVSSETNV (SEQ ID NO: 42);

7) antibody ZIL94: VH-CDR1 of TYFMS (SEQ ID NO: 43), VH-CDR2 ofLISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 of FWRAFND (SEQ ID NO: 45),VL-CDR1 of GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 of DTGSRPS (SEQ IDNO: 47), VL-CDR3 of SLYTDSDILV (SEQ ID NO: 48);

8) antibody ZIL154: VH-CDR1 of DRGMS (SEQ ID NO: 49), VH-CDR2 ofYIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 of WDGSSFDY (SEQ ID NO: 51),VL-CDR1 of KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 of KVSNRDP (SEQ IDNO: 53), VL-CDR3 of MQAIHFPLT (SEQ ID NO: 54);

9) antibody ZIL159: VH-CDR1 of SYVMT (SEQ ID NO: 55), VH-CDR2 ofGINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 of GDIVATGTSY (SEQ ID NO:57), VL-CDR1 of SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 of KDTERPS (SEQ IDNO: 59), VL-CDR3 of KSAVSIDVGV (SEQ ID NO: 60);

10) antibody ZIL171: VH-CDR1 of TYVMN (SEQ ID NO: 61), VH-CDR2 ofSINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 of SMVGPFDY (SEQ ID NO: 63),VL-CDR1 of SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 of KDTERPS (SEQ ID NO:65), VL-CDR3 of ESAVSSDTIV (SEQ ID NO: 66); or

11) a variant of 1) to 10) that differs from respective parent antibody15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, or ZIL171by addition, deletion, and/or substitution of one or more amino acidresidues in at least one of VH or VL CDR1, CDR2, or CDR3.

In one embodiment, the IL-31 mimotope used in the vaccine compositionbinds to an antibody which specifically binds feline IL-31, wherein theantibody binds to a region between about amino acid residues 125 and 134of a feline IL-31 sequence represented by SEQ ID NO: 157(Feline_IL31_wildtype). In some embodiments, such an antibody includes aVL chain comprising Framework 2 (FW2) changes selected from thefollowing: an Asparagine in place of Lysine at position 42, anIsoleucine in place of Valine at position 43, a Valine in place ofLeucine at position 46, an Asparagine in place of Lysine at position 49,and combinations thereof, wherein the positions are in reference to thenumbering of SEQ ID NO: 127 (FEL_15H05_VL1).

In some embodiments, the mimotope binds to an antibody characterized inthat:

-   -   1) antibody ZIL1 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL1_VL: (SEQ ID NO: 77)QSVLTQPTSVSGSLGQRVTISCSGSTNNIGILAATWYQQLPGKAPKVLVYSDGNRPSGVPDRFSGSKSGNSATLTITGLQAEDEADYYCQSFDTTLDA YVFGSGTQLTVL, andb) a variable heavy chain comprising CAN-ZIL1_VH: (SEQ ID NO: 75)EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYGMSWVRQAPGKGLQWVAHINSGGSSTYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVEVYTTLAAFWTDNFDYWGQGTLVTVSS;

-   -   2) antibody ZIL8 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL8_VL: (SEQ ID NO: 81)QSVLTQPASVSGSLGQKVTISCTGSSSNIGSGYVGWYQQLPGTGPRTLIYYNSDRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSVYDRTFNA VFGGGT, andb) a variable heavy chain comprising CAN-ZIL8_VH: (SEQ ID NO: 79)EVQLVESGGDLVKPAGSLRLSCVASGFTFSDYAMSWVRQAPGRGLQWVAGIDSVGSGTSYADAVKGRFTISRDDAKNTLYLQMFNLRAEDTAIYYCAS GFPGSFEHWGQGTLVTVSS;

-   -   3) antibody ZIL9 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL9_VL: (SEQ ID NO: 85)SSVLTQPPSVSVSLGQTATISCSGESLNEYYTQWFQQKAGQAPVLVIYRDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVDTGTLVF GGGTHLAVL, andb) a variable heavy chain comprising CAN-ZIL9_VH: (SEQ ID NO: 83)EVQLVESGGDLVKPPGSLRLSCVASGFTFSSYDMTWVRQAPGKGLQWVADVNSGGTGTAYAVAVKGRFTISRDNAKKTLYLQMNSLRAEDTAVYYCAK LGVRDGLSVWGQGTLVTVSS;

-   -   4) antibody ZIL11 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL11_VL: (SEQ ID NO: 89)SSVLTQPPSVSVSLGQTATISCSGESLSNYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVSSDTIVF GGGT, andb) a variable heavy chain comprising CAN-ZIL11_VH: (SEQ ID NO: 87)EVQLVESGGDLVKPAGSLRLSCVASGFTFRTYVMNWVRQAPGKGLQWVASINGGGSSPTYADAVRGRFTVSRDNAQNSLFLQMNSLRAEDTAVYFCVV SMVGPFDYWGQGTLVTVSS;

-   -   5) antibody ZIL69 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL69_VL: (SEQ ID NO: 93)SSVLTQPPSVSVSLGQTATISCSGESLNKYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSAGNTHTLTISGARAEDEADYYCESAVSSETNVF GSGTQLTVL, andb) a variable heavy chain comprising CAN-ZIL69_VH: (SEQ ID NO: 91)EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYAMKWVRQAPGKGLQWVATINNDGTRTGYADAVRGRFTISKDNAKNTLYLQMDSLRADDTAVYYCTKGNAESGCTGDHCPPYWGQGTLVTVSS;

-   -   6) antibody ZIL94 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL94_VL: (SEQ ID NO: 97)QTVVIQEPSLSVSPGGTVTLTCGLNSGSVSTSNYPGWYQQTRGRTPRTIIYDTGSRPSGVPNRFSGSISGNKAALTITGAQPEDEADYYCSLYTDSDI LVFGGGTHLTVL, andb) a variable heavy chain comprising CAN-ZIL94_VH: (SEQ ID NO: 95)EVQLVDSGGDLVKPGGSLRLSCVASGFTFSTYFMSWVRQAPGRGLQWVALISSDGSGTYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCAI FWRAFNDWGQGTLVTVSS;

-   -   7) antibody ZIL154 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL154_VL: (SEQ ID NO: 101)DIVVTQTPLSLSVSPGETASFSCKASQSLLHSDGNTYLDWFRQKPGQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLRISGVEADDAGLYYCMQAIH FPLTFGAGTKVELK, andb) a variable heavy chain comprising CAN-ZIL154_VH: (SEQ ID NO: 99)EVHLVESGGDLVKPWGSLRLSCVASGFTFSDRGMSWVRQSPGKGLQWVAYIRYDGSRTDYADAVEGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR WDGSSFDYWGQGTLVTVSS;

-   -   8) antibody ZIL159 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL159_VL: (SEQ ID NO: 105)SNVLTQPPSVSVSLGQTATISCSGETLNRFYTQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNIHTLTISGARAEDEAAYYCKSAVSIDVGVF GGGTHLTVF, andb) a variable heavy chain comprising CAN-ZIL159_VH: (SEQ ID NO: 103)EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYVMTWVRQAPGKGLQWVAGINSEGSRTAYADAVKGRFTISRDNAKNTLYLQIDSLRAEDTAIYYCATGDIVATGTSYWGQGTLVTVSS;  and

-   -   9) antibody ZIL171 includes at least one of the following:

a) a variable light chain comprising CAN-ZIL171_VL: (SEQ ID NO: 109)SSVLTQPPSVSVSLGQTATISCSGKSLSYYYAQWFQQKAGQAPVLVIYKDTERPSGIPDRFSGSSSGNTHTLTISGARAEDEADYYCESAVSSDTIVF GGGTHLTVL, andb) a variable heavy chain comprising CAN-ZIL171_VH: (SEQ ID NO: 107)EVQLVESGGDLVKPAGSLRLSCVASGFTFRTYVMNWVRQAPGKGLQWVASINGGGSSPTYADAVRGRFTVSRDNAQNSLFLQMNSLRAEDTAIYFCVV SMVGPFDYWGHGTLVTVSS.

A host cell can be used to produce an antibody described above. Suchantibodies can be used in the diagnostic procedures described as part ofthis invention, although the diagnostic procedures are not limited tothese particular antibodies.

Nucleotide sequences encoding the variable regions of the light andheavy chains of the anti-IL-31 antibody can be employed to make theanti-IL-31 antibodies described herein. Such nucleotide sequencesinclude, but are not limited to, any nucleotide sequence that encodesthe amino acid sequence of the15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69,ZIL94, ZIL154, ZIL159 or ZIL171 antibodies or IL-31-specificpolypeptides or peptides thereof. In addition, in one embodiment,nucleotide sequences encoding the IL-31 mimotopes can be used torecombinantly produce the mimotopes alone or as part of a fusion proteintogether with the carrier polypeptide. Alternatively, or in addition,the mimotopes can be chemically synthesized.

In some embodiments, an isolated nucleic acid can be employed to make auseful antibody (such as that used in one of the diagnostic methodsdescribed herein), wherein the nucleic acid sequence encodes at leastone of the following combinations of variable heavy complementarydetermining region (CDR) sequences:

-   -   1) 15H05: variable heavy (VH)-CDR1 of SYTIH (SEQ ID NO: 1),        VH-CDR2 of NINPTSGYTENNQRFKD (SEQ ID NO: 2), and VH-CDR3 of        WGFKYDGEWSFDV (SEQ ID NO: 3);    -   2) ZIL1: VH-CDR1 of SYGMS (SEQ ID NO: 13), VH-CDR2 of        HINSGGSSTYYADAVKG (SEQ ID NO:14), and VH-CDR3 of VYTTLAAFWTDNFDY        (SEQ ID NO: 15);    -   3) ZIL8: VH-CDR1 of DYAMS (SEQ ID NO: 19), VH-CDR2 of        GIDSVGSGTSYADAVKG (SEQ ID NO: 20), and VH-CDR3 of GFPGSFEH (SEQ        ID NO: 21);    -   4) ZIL9: VH-CDR1 of SYDMT (SEQ ID NO: 25), VH-CDR2 of        DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), and VH-CDR3 of LGVRDGLSV (SEQ        ID NO: 27);    -   5) ZIL11: VH-CDR1 of TYVMN (SEQ ID NO: 31), VH-CDR2 of        SINGGGSSPTYADAVRG (SEQ ID NO: 32), and VH-CDR3 of SMVGPFDY (SEQ        ID NO: 33);    -   6) ZIL69: VH-CDR1 of SYAMK (SEQ ID NO: 37), VH-CDR2 of        TINNDGTRTGYADAVRG (SEQ ID NO: 38), and VH-CDR3 of        GNAESGCTGDHCPPY (SEQ ID NO: 39);    -   7) ZIL94: VH-CDR1 of TYFMS (SEQ ID NO: 43), VH-CDR2 of        LISSDGSGTYYADAVKG (SEQ ID NO: 44), and VH-CDR3 of FWRAFND (SEQ        ID NO: 45)    -   8) ZIL154: VH-CDR1 of DRGMS (SEQ ID NO: 49), VH-CDR2 of        YIRYDGSRTDYADAVEG (SEQ ID NO: 50), and VH-CDR3 of WDGSSFDY (SEQ        ID NO: 51);    -   9) ZIL159: VH-CDR1 of SYVMT (SEQ ID NO: 55), VH-CDR2 of        GINSEGSRTAYADAVKG (SEQ ID NO: 56), and VH-CDR3 of GDIVATGTSY        (SEQ ID NO: 57);    -   10) ZIL171: VH-CDR1 of TYVMN (SEQ ID NO: 61), VH-CDR2 of

SINGGGSSPTYADAVRG (SEQ ID NO: 62), and VH-CDR3 of SMVGPFDY (SEQ ID NO:63), or

-   -   11) a variant of 1) to 10) that differs from the CDRs of        respective parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11,        ZIL69, ZIL94, ZIL154, ZIL159, or ZIL171 by addition, deletion,        and/or substitution of one or more amino acid residues in at        least one of VH CDR1, CDR2, or CDR3.

In another embodiment, the isolated nucleic acid comprises a nucleicacid sequence encoding at least one of the following combinations ofvariable light complementary determining region (CDR) sequences:

-   -   1) 15H05: variable light (VL)-CDR1 of RASQGISIWLS (SEQ ID NO:        4), VL-CDR2 of KASNLHI (SEQ ID NO: 5), and VL-CDR3 of LQSQTYPLT        (SEQ ID NO: 6);    -   2) ZIL1: VL-CDR1 of SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 of        SDGNRPS (SEQ ID NO: 17, and VL-CDR3 of QSFDTTLDAYV (SEQ ID        NO:18);    -   3) ZIL8: VL-CDR1 of TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 of        YNSDRPS (SEQ ID NO: 23), and VL-CDR3 of SVYDRTFNAV (SEQ ID NO:        24);    -   4) ZIL9: VL-CDR1 of SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 of        RDTERPS (SEQ ID NO: 29), and VL-CDR3 of ESAVDTGTLV (SEQ ID NO:        30);    -   5) ZIL11: VL-CDR1 of SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 of        KDTERPS (SEQ ID NO: 35), and VL-CDR3 of ESAVSSDTIV (SEQ ID NO:        36);    -   6) ZIL69: VL-CDR1 of SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 of        KDTERPS (SEQ ID NO: 41), and VL-CDR3 of ESAVSSETNV (SEQ ID NO:        42);    -   7) ZIL94: VL-CDR1 of GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 of        DTGSRPS (SEQ ID NO: 47), and VL-CDR3 of SLYTDSDILV (SEQ ID NO:        48);    -   8) ZIL154: VL-CDR1 of KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2        of KVSNRDP (SEQ ID NO: 53), and VL-CDR3 of MQAIHFPLT (SEQ ID NO:        54);    -   9) ZIL159: VL-CDR1 of SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 of        KDTERPS (SEQ ID NO: 59), and VL-CDR3 of KSAVSIDVGV (SEQ ID NO:        60);    -   10) ZIL171: VL-CDR1 of SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 of        KDTERPS (SEQ ID NO: 65), and VL-CDR3 of ESAVSSDTIV (SEQ ID NO:        66); or    -   11) a variant of 1) to 10) that differs from the CDRs of        respective parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11,        ZIL69, ZIL94, ZIL154, ZIL159, or ZIL171 by addition, deletion,        and/or substitution of one or more amino acid residues in at        least one of VL CDR1, CDR2, or CDR3.

In yet another embodiment, the isolated nucleic acid used in themanufacture of an antibody described herein comprises a nucleic acidsequence encoding the above-described variable light complementarydetermining region (CDR) sequences, as well as the above-describedvariable heavy CDR sequences of respective parent antibody 15H05, ZIL1,ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, or ZIL171 or variantsthereof.

A vector including at least one of the nucleic acids described above canbe used in the manufacture of these antibodies. As will be described infurther detail below, the nucleic acid sequence encoding at least one ofthe above-described combinations of variable heavy complementarydetermining region (CDR) sequences may be contained on the same vectortogether with the nucleic acid sequence encoding at least one of theabove-described combinations of variable light CDR sequences.Alternatively, the nucleic acid sequence encoding at least one of theabove-described combinations of variable light CDR sequences and thenucleic acid sequence encoding at least one of the above-describedcombinations of variable heavy CDR sequences may each be contained onseparate vectors.

Because the genetic code is degenerate, more than one codon can be usedto encode a particular amino acid. Using the genetic code, one or moredifferent nucleotide sequences can be identified, each of which would becapable of encoding the amino acid. The probability that a particularoligonucleotide will, in fact, constitute the actual XXX-encodingsequence can be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic orprokaryotic cells expressing an anti-IL-31 antibody or IL-31-specificportion thereof. Such “codon usage rules” are disclosed by Lathe, etal., 183 J. Molec. Biol. 1-12 (1985). Using the “codon usage rules” ofLathe, a single nucleotide sequence, or a set of nucleotide sequencesthat contains a theoretical “most probable” nucleotide sequence capableof encoding anti-IL-31 sequences can be identified. It is also intendedthat the antibody coding regions could also be provided by alteringexisting antibody genes using standard molecular biological techniquesthat result in variants (agonists) of the antibodies and peptidesdescribed herein. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of theanti-IL-31 antibodies or IL-31-specific polypeptides or peptides (suchas antibody portions or fragments). Also, variants of the peptidemimotopes described herein can be made by altering the nucleotidesequence encoding the parent peptide mimotope.

For example, one class of substitutions is conservative amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid in an anti-IL-31antibody, or IL-31 specific polypeptide orpeptide by another amino acid of like characteristics. Likewise, IL-31mimotopes which bind to such an antibody or an antigen-binding portionthereof can include conservative substitutions or other types of aminoacid substitutions. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu, substitution between the amideresidues Asn and Gln, exchange of the basic residues Lys and Arg,replacements among the aromatic residues Phe, Tyr, and the like.Guidance concerning which amino acid changes are likely to bephenotypically silent is found in Bowie et al., 247 Science 1306-10(1990).

Variant or agonist anti-IL-31 antibodies or IL-31-specific polypeptides,or peptides may be fully functional or may lack function in one or moreactivities. Likewise, variant or agonist IL-31 mimotopes may be fullyfunctional or may lack function in one or more activities. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as epitope binding or in vitro ADCC activity. Sites thatare critical for ligand-receptor binding can also be determined bystructural analysis such as crystallography, nuclear magnetic resonance,or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904(1992); de Vos et al., 255 Science 306-12 (1992).

Moreover, polypeptides often contain amino acids other than the twenty“naturally occurring” amino acids. Further, many amino acids, includingthe terminal amino acids, may be modified by natural processes, such asprocessing and other post-translational modifications, or by chemicalmodification techniques well known in the art. Known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties (2nd ed., T. E.Creighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews areavailable on this subject, such as by Wold, Posttranslational CovalentModification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983);Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663Ann. NY Acad. Sci. 48-62 (1992).

Accordingly, the IL-31-specific antibodies, polypeptides, and peptidesdescribed herein, as well as the IL-31 peptide mimotopes describedherein also encompass derivatives or analogs in which a substitutedamino acid residue is not one encoded by the genetic code.

Similarly, the additions and substitutions in the amino acid sequence aswell as variations, and modifications just described may be equallyapplicable to the amino acid sequence of the IL-31 antigen and/orepitope or peptides thereof, and are thus encompassed by the presentinvention.

Antibody and Mimotope Derivatives

Included within the scope of this invention are antibody and mimotopederivatives. A “derivative” of an antibody or mimotope containsadditional chemical moieties not normally a part of the protein orpeptide. Covalent modifications of the protein or peptide are includedwithin the scope of this invention. Such modifications may be introducedinto the molecule by reacting targeted amino acid residues of theantibody or mimotope with an organic derivatizing agent that is capableof reacting with selected side chains or terminal residues. For example,derivatization with bifunctional agents, well-known in the art, isuseful for cross-linking the antibody or fragment or mimotope to awater-insoluble support matrix or to other macromolecular carriers.

Derivatives also include radioactively labeled monoclonal antibodies ormimotopes. For example, with radioactive iodine (¹²⁵I,¹³¹I), carbon(¹⁴C), sulfur (³⁵S), indium (¹¹¹In), tritium (³H) or the like;conjugates of monoclonal antibodies with biotin or avidin, with enzymes,such as horseradish peroxidase, alkaline phosphatase,beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acidanhydrase, acetylcholine esterase, lysozyme, malate dehydrogenase orglucose 6-phosphate dehydrogenase; and also conjugates of monoclonalantibodies with bioluminescent agents (such as luciferase),chemoluminescent agents (such as acridine esters) or fluorescent agents(such as phycobiliproteins). Likewise, the mimotopes can be labeled insome embodiments.

Another derivative bifunctional antibody is a bispecific antibody,generated by combining parts of two separate antibodies that recognizetwo different antigenic groups. This may be achieved by crosslinking orrecombinant techniques.

Additionally, moieties may be added to the antibody or a portion thereofor to the IL-31 mimotopes described herein to increase half-life in vivo(e.g., by lengthening the time to clearance from the blood stream. Suchtechniques include, for example, adding PEG moieties (also termedPEGylation), and are well-known in the art. See U.S. Patent. Appl. Pub.No. 20030031671.

Recombinant Expression of Antibodies, Mimotopes, and CarrierPolypeptides

In some embodiments, the nucleic acids encoding a subject monoclonalantibody or a fusion protein containing both the mimotope and thecarrier polypeptide are introduced directly into a host cell, and thecell is incubated under conditions sufficient to induce expression ofthe encoded antibody or fusion protein. After the subject nucleic acidshave been introduced into a cell, the cell is typically incubated,normally at 37° C., sometimes under selection, for a period of about1-24 hours in order to allow for the expression of the antibody orfusion protein carrying the peptide mimotope and carrier polypeptide. Inone embodiment, the antibody or fusion protein secreted into thesupernatant of the media in which the cell is growing.

Traditionally, monoclonal antibodies have been produced as nativemolecules in murine hybridoma lines. In addition to that technology, thepresent invention provides for recombinant DNA expression of monoclonalantibodies. This allows the production of caninized, felinized,equinized, humanized, fully canine, fully feline, fully equine, andfully human antibodies, as well as a spectrum of antibody derivativesand fusion proteins in a host species of choice.

A nucleic acid sequence encoding at least one anti-IL-31 antibody,portion or IL-31-specific polypeptide thereof or a nucleic acid sequenceencoding as part thereof at least one IL-31 peptide mimotope which bindsto such an antibody or portion thereof may be recombined with vector DNAin accordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, e.g., by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL,(Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al.1993 supra, may be used to construct nucleic acid sequences which encodea monoclonal antibody molecule or antigen binding region thereof, orIL-31 peptide mimotope.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as anti-IL-31peptides or antibody portions, or as fusion proteins carrying IL-31mimotopes in recoverable amounts. The precise nature of the regulatoryregions needed for gene expression may vary from organism to organism,as is well known in the analogous art. See, e.g., Sambrook et al., 2001supra; Ausubel et al., 1993 supra.

The present invention accordingly encompasses the expression of ananti-IL-31 antibody or IL-31-specific polypeptide or peptide, or fusionprotein including an IL-31 mimotope, in either prokaryotic or eukaryoticcells. Suitable hosts include bacterial or eukaryotic hosts includingbacteria, yeast, insects, fungi, bird and mammalian cells either invivo, or in situ, or host cells of mammalian, insect, bird or yeastorigin. The mammalian cell or tissue may be of human, primate, hamster,rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but anyother mammalian cell may be used.

In one embodiment, the introduced nucleotide sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. See, e.g., Ausubel et al., 1993 supra.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Example prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli (such as, for example, pBR322,ColE1, pSC101, pACYC 184, .pi.VX). Such plasmids are, for example,disclosed by Maniatis et al., 1989 supra; Ausubel et al, 1993 supra.Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids aredisclosed by Gryczan, in THE MOLEC. BIO. OF THE BACILLI 307-329(Academic Press, NY, 1982). Suitable Streptomyces plasmids includepIJ101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)), andStreptomyces bacteriophages such as .phi.C31 (Chater et al., in SIXTHINT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido,Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John etal., 8 Rev. Infect. Dis. 693-704 (1986); Izaki, 33 Jpn. J. Bacteriol.729-42 (1978); and Ausubel et al., 1993 supra.

Alternatively, gene expression elements useful for the expression ofcDNA encoding anti-IL-31 antibodies or peptides, or fusion proteins asdescribed herein include, but are not limited to (a) viral transcriptionpromoters and their enhancer elements, such as the SV40 early promoter(Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR(Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982)), and Moloneymurine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b)splice regions and polyadenylation sites such as those derived from theSV40 late region (Okayarea et al., MCB, 3: 280 (1983), and (c)polyadenylation sites such as in SV40 (Okayama et al., 1983, supra).

Immunoglobulin cDNA genes can be expressed as described by Weidle etal., 51(1) Gene 21-29 (1987), using as expression elements the SV40early promoter and its enhancer, the mouse immunoglobulin H chainpromoter enhancers, SV40 late region mRNA splicing, rabbit S-globinintervening sequence, immunoglobulin and rabbit S-globin polyadenylationsites, and SV40 polyadenylation elements.

For immunoglobulin genes comprised of part cDNA, part genomic DNA(Whittle et al., 1 Protein Engin. 499-505 (1987)), the transcriptionalpromoter can be human cytomegalovirus, the promoter enhancers can becytomegalovirus and mouse/human immunoglobulin, and mRNA splicing andpolyadenylation regions can be the native chromosomal immunoglobulinsequences.

In one embodiment, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enhancers are either or both the mouse immunoglobulin heavychain enhancer and the viral LTR enhancer, the splice region contains anintron of greater than 31 bp, and the polyadenylation and transcriptiontermination regions are derived from the native chromosomal sequencecorresponding to the immunoglobulin chain being synthesized. In otherembodiments, cDNA sequences encoding other proteins are combined withthe above-recited expression elements to achieve expression of theproteins in mammalian cells.

Each fused gene can be assembled in, or inserted into, an expressionvector. Recipient cells capable of expressing the chimericimmunoglobulin chain gene product are then transfected singly with ananti-IL-31 peptide or chimeric H or chimeric L chain-encoding gene, orare co-transfected with a chimeric H and a chimeric L chain gene. Thetransfected recipient cells are cultured under conditions that permitexpression of the incorporated genes and the expressed immunoglobulinchains or intact antibodies or fragments are recovered from the culture.

In one embodiment, the fused genes encoding the anti-IL-31 peptide orchimeric H and L chains, or portions thereof are assembled in separateexpression vectors that are then used to co-transfect a recipient cell.Alternatively the fused genes encoding the chimeric H and L chains canbe assembled on the same expression vector.

For transfection of the expression vectors and production of thechimeric antibody, the recipient cell line may be a myeloma cell.Myeloma cells can synthesize, assemble and secrete immunoglobulinsencoded by transfected immunoglobulin genes and possess the mechanismfor glycosylation of the immunoglobulin. Myeloma cells can be grown inculture or in the peritoneal cavity of a mouse, where secretedimmunoglobulin can be obtained from ascites fluid. Other suitablerecipient cells include lymphoid cells such as B lymphocytes of human ornon-human origin, hybridoma cells of human or non-human origin, orinterspecies heterohybridoma cells.

The expression vector carrying a nucleotide sequence encoding chimeric,caninized, felinized, equinized, humanized, fully canine, fully feline,fully equine, or fully human anti-IL-31 antibody construct sequences oran IL-31-specific polypeptide or peptide (e.g., antigen-binding portionof the antibodies described herein), or an expression vector carrying anucleotide sequence encoding a fusion protein as described herein, canbe introduced into an appropriate host cell by any of a variety ofsuitable means, including such biochemical means as transformation,transfection, conjugation, protoplast fusion, calciumphosphate-precipitation, and application with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment.Johnston et al., 240 Science 1538-1541 (1988).

Yeast can provide substantial advantages over bacteria for theproduction of immunoglobulin H and L chains. Yeasts carry outpost-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies now exist which utilize strongpromoter sequences and high copy number plasmids which can be used forproduction of the desired proteins in yeast. Yeast recognizes leadersequences of cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides). Hitzman et al., 11th IntlConference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

Yeast gene expression systems can be routinely evaluated for the levelsof production, secretion and the stability of anti-IL-31 peptides,antibody and assembled murine and chimeric, heterochimeric, caninized,felinized, equinized, humanized, fully canine, fully feline, fullyequine, or fully human antibodies, fragments and regions thereof. Any ofa series of yeast gene expression systems incorporating promoter andtermination elements from the actively expressed genes coding forglycolytic enzymes produced in large quantities when yeasts are grown inmedia rich in glucose can be utilized. Known glycolytic genes can alsoprovide very efficient transcription control signals. For example, thepromoter and terminator signals of the phosphoglycerate kinase (PGK)gene can be utilized. A number of approaches can be taken for evaluatingoptimal expression plasmids for the expression of cloned immunoglobulincDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRLPress, Oxford, UK 1985).

Bacterial strains can also be utilized as hosts for the production ofantibody molecules or peptides, or fusion proteins described by thisinvention. Plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with a host cell are used inconnection with these bacterial hosts. The vector carries a replicationsite, as well as specific genes which are capable of providingphenotypic selection in transformed cells. A number of approaches can betaken for evaluating the expression plasmids for the production ofmurine, chimeric, heterochimeric, caninized, felinized, equinized,humanized, fully canine, fully feline, fully equine, or fully humanantibodies, fragments and regions or antibody chains encoded by thecloned immunoglobulin cDNAs in bacteria (see Glover, 1985 supra;Ausubel, 1993 supra; Sambrook, 2001 supra; Colligan et al., eds. CurrentProtocols in Immunology, John Wiley & Sons, NY, N.Y. (1994-2001);Colligan et al., eds. Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y. (1997-2001).

Host mammalian cells may be grown in vitro or in vivo. Mammalian cellsprovide post-translational modifications to immunoglobulin proteinmolecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the antibody molecules, and secretion offunctional antibody protein.

Mammalian cells which can be useful as hosts for the production ofantibody proteins, in addition to the cells of lymphoid origin describedabove, include cells of fibroblast origin, such as Vero (ATCC CRL 81) orCHO-K1 (ATCC CRL 61) cells.

Many vector systems are available for the expression of clonedanti-IL-31 peptides H and L chain genes in mammalian cells (see Glover,1985 supra). Different approaches can be followed to obtain completeH₂L₂ antibodies. It is possible to co-express H and L chains in the samecells to achieve intracellular association and linkage of H and L chainsinto complete tetrameric H₂L₂ antibodies and/or anti-IL-31 peptides. Theco-expression can occur by using either the same or different plasmidsin the same host. Genes for both H and L chains and/or anti-IL-31peptides can be placed into the same plasmid, which is then transfectedinto cells, thereby selecting directly for cells that express bothchains. Alternatively, cells can be transfected first with a plasmidencoding one chain, for example the L chain, followed by transfection ofthe resulting cell line with an H chain plasmid containing a secondselectable marker. Cell lines producing anti-IL-31 peptides and/or H₂L₂molecules via either route could be transfected with plasmids encodingadditional copies of peptides, H, L, or H plus L chains in conjunctionwith additional selectable markers to generate cell lines with enhancedproperties, such as higher production of assembled H₂L₂ antibodymolecules or enhanced stability of the transfected cell lines.

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds/components that interact directly or indirectly with theantibody molecule.

Once an antibody has been produced, it may be purified by any methodknown in the art for purification of an immunoglobulin molecule, forexample, by chromatography (e.g., ion exchange, affinity, particularlyaffinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. In manyembodiments, antibodies are secreted from the cell into culture mediumand harvested from the culture medium.

Pharmaceutical Applications

The vaccine compositions of the present invention can be used forexample in the treatment of and/or protection against IL-31-mediateddisorders, such as pruritic and/or allergic conditions in mammals, suchas dogs, cats, horses, and humans. The pharmaceutical compositions ofthis invention are useful for parenteral administration, e.g.,subcutaneously, intramuscularly or intravenously. Other suitable modesof administration are described herein.

The vaccines of the present invention can be administered either asindividual therapeutic agents or in combination with other therapeuticagents. They can be administered alone, but are generally administeredwith a pharmaceutical carrier selected on the basis of the chosen routeof administration and standard pharmaceutical practice.

Administration of the vaccine compositions disclosed herein may becarried out by any suitable means, including parenteral injection (suchas intraperitoneal, subcutaneous, or intramuscular injection), orally,or by topical administration of the vaccines to an airway surface.Topical administration to an airway surface can be carried out byintranasal administration (e.g., by use of dropper, swab, or inhaler).Topical administration of the vaccines to an airway surface can also becarried out by inhalation administration, such as by creating respirableparticles of a pharmaceutical formulation (including both solid andliquid particles) containing the vaccines as an aerosol suspension, andthen causing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well known, and any conventional technique can beemployed. Oral administration may be, for example, in the form of aningestable liquid or solid formulation.

In some desired embodiments, the vaccines are administered by parenteralinjection. For parenteral administration, the vaccines can be formulatedas a solution, suspension, emulsion or lyophilized powder in associationwith a pharmaceutically acceptable parenteral vehicle. For example thevehicle may be a solution of the combination of the mimotope and thecarrier polypeptide (e.g., mimotope conjugate) or a cocktail thereofdissolved in an acceptable carrier, such as an aqueous carrier suchvehicles are water, saline, Ringer's solution, dextrose solution,trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3%glycine and the like. Liposomes and nonaqueous vehicles such as fixedoils can also be used. These solutions are sterile and generally free ofparticulate matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjustment agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. Also, as described herein, the vaccine compositions ofthis invention include an adjuvant or adjuvant formulation. Theconcentration of mimotope conjugate in these vaccine compositions canvary widely, for example from less than about 0.5%, usually at or atleast about 1% to as much as 15% or 20% by weight and will be selectedprimarily based on fluid volumes, viscosities, etc., in accordance withthe particular mode of administration selected. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by commonly usedtechniques.

Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in, for example, REMINGTON'S PHARMA. SCI. (15th ed., MackPub. Co., Easton, Pa., 1980).

The vaccines of this invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. Any suitablelyophilization and reconstitution techniques can be employed. It will beappreciated by those skilled in the art that lyophilization andreconstitution can lead to varying degrees of activity loss and that uselevels may have to be adjusted to compensate.

The compositions containing the present IL-31 mimotopes (e.g., IL-31mimotope conjugates) or a cocktail thereof can be administered forprevention of recurrence and/or therapeutic treatments for existingdisease. Suitable pharmaceutical carriers are described in the mostrecent edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standardreference text in this field of art.

In therapeutic application, compositions are administered to a subjectalready suffering from a disease, in an amount sufficient to cure or atleast partially arrest or alleviate the disease and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose” or a “therapeutically effective amount”. Amountseffective for this use will depend upon the severity of the disease andthe general state of the subject's own immune system. A therapeuticallyeffective amount of a vaccine composition according to the invention maybe readily determined by one of ordinary skill in the art.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms kind of concurrenttreatment, frequency of treatment, and the effect desired.

As a non-limiting example, treatment of IL-31-related pathologies indogs, cats, horses, or humans can be provided as a biweekly or monthlydosage of vaccines of the present invention in the dosage rangedescribed above.

Single or multiple administrations of the vaccine compositions can becarried out with dose levels and pattern being selected by the treatingveterinarian or physician. In any event, the pharmaceutical formulationsshould provide a quantity of the vaccine compositions of this inventionsufficient to effectively treat the subject.

Diagnostic Applications

The present invention also provides the IL-31 mimotopes and anti-IL-31antibodies for use in diagnostic methods for detecting IL-31 oranti-IL-31 antibodies in mammalian samples, including, but not limitedto, samples from mammals known to be or suspected of having a puriticand/or allergic condition.

For example, the present invention provides a method of determining theidentity and/or amount of an anti-IL-31 antibody in a sample. Thismethod includes incubating a sample including an anti-IL-31 antibodywith at least one IL-31 mimotope such as a feline IL-31 mimotope, acanine IL-31 mimotope, a horse IL-31 mimotope, or a human IL-31mimotope; and determining the identity and/or quantity of the anti-IL-31in the sample.

In one embodiment, the canine IL-31 mimotope employed in the method todetermine the identity and/or amount of an anti-IL-31 antibody in thesample is and/or comprises as part thereof the amino acid sequenceSVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187),NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192),APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) orvariants thereof that retain anti-IL-31 binding.

In another embodiment, the feline IL-31 mimotope employed in such amethod is and/or comprises as part thereof the amino acid sequenceSMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ IDNO: 193), APAHRLQPSDIRKIILELRPM SKG (SEQ ID NO: 197), IGLPES (SEQ ID NO:201) or variants thereof that retain anti-IL-31 binding.

In a further embodiment, the equine IL-31 mimotope employed in such amethod is and/or comprises as part thereof the amino acid sequenceSMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ IDNO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO:202) or variants thereof that retain anti-IL-31 binding.

In a still further, the human IL-31 mimotope employed in such a methodis and/or comprises as part thereof the amino acid sequence SVPTDTHECKRF(SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191),HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195),LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203) orvariants thereof that retain anti-IL-31 binding.

In one embodiment of the above-described diagnostic method, the mimotopeis a capture reagent bound to a solid surface. In one embodiment, thesample is added to the mimotope capture reagent; and secondary detectionreagents are then added to quantify the amount of the antibody in thesample.

The present invention also provides a method of determining the amountof IL-31 in a sample from a mammal. Such a method will have utility fordetecting IL-31 from multiple species. Such a method includes incubatinga mammalian sample comprising IL-31 with a labeled anti-IL-31 antibody:IL-31 mimotope complex tethered to a solid surface, wherein the mimotopein the complex is selected from the group consisting of a feline IL-31mimotope, a canine IL-31 mimotope, a horse IL-31 mimotope, and a humanIL-31 mimotope; and determining the level of the IL-31 in the sample,wherein the labeled anti-IL-31 antibody in the complex has an affinityto the mimotope in the complex that is lower than its affinity to theIL-31 in the sample. In one embodiment of this method, the determiningstep comprises measuring the signal coming from labeled antibody whichis liberated from the solid surface when the IL-31 in the sample bindsto the labeled anti-IL-3 antibody of the complex, the level of IL-31 inthe sample being inversely proportional to the signal.

In one embodiment, the canine IL-31 mimotope employed in the method ofdetermining the amount of IL-31 in the sample is and/or comprises aspart thereof the amino acid sequence SVPADTFECKSF (SEQ ID NO: 186),SVPADTFERKSF (SEQ ID NO: 187), NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ IDNO: 192), APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO:200) or variants thereof that retain anti-IL-31 binding.

In another embodiment, the feline IL-31 mimotope employed in such amethod is and/or comprises as part thereof the amino acid sequenceSMPADNFERKNF (SEQ ID NO: 188), NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ IDNO: 193), APAHRLQPSDIRKIILELRPMSKG (SEQ ID NO: 197), IGLPES (SEQ ID NO:201) or variants thereof that retain anti-IL-31 binding.

In yet another embodiment, the equine IL-31 mimotope employed in such amethod is and/or comprises as part thereof the amino acid sequenceSMPTDNFERKRF (SEQ ID NO: 189), NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ IDNO: 194), GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO:202) or variants thereof that retain anti-IL-31 binding.

In a still further embodiment, the human IL-31 mimotope employed in sucha method is and/or comprises as part thereof the amino acid sequenceSVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQ ID NO: 191),HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195),LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203) orvariants thereof that retain anti-IL-31 binding.

In some embodiments of any of the diagnostic methods of the invention,the mimotope binds to an anti-IL31 antibody or antigen-binding portionthereof that specifically binds to a region on a mammalian IL-31 proteininvolved with interaction of the IL-31 protein with its co-receptor. Inone embodiment of the diagnostic methods of this invention, the bindingof said antibody to said region is impacted by mutations in a 15H05epitope binding region selected from the group consisting of:

-   -   a) a region between about amino acid residues 124 and 135 of a        feline IL-31 sequence represented by SEQ ID NO: 157        (Feline_IL31_wildtype);    -   b) a region between about amino acid residues 124 and 135 of a        canine IL-31 sequence represented by SEQ ID NO: 155        (Canine_IL31); and    -   c) a region between about amino acid residues 118 and 129 of an        equine IL-31 sequence represented by SEQ ID NO: 165        (Equine_IL31).

In one embodiment of the diagnostic methods of the present invention themimotope binds to an anti-IL31 antibody or antigen-binding portionthereof that specifically binds to the aforementioned 15H05 epitoperegion. In one specific embodiment of any of the diagnostic methods ofthe instant invention, the mimotope binds to an anti-IL-31 antibody orantigen-binding portion thereof comprising at least one of the followingcombinations of complementary determining region (CDR) sequences:

-   -   1) antibody 15H05: variable heavy (VH)-CDR1 of SYTIH (SEQ ID NO:        1), VH-CDR2 of NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 of        WGFKYDGEWSFDV (SEQ ID NO: 3), variable light (VL)-CDR1 of        RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 of KASNLHI (SEQ ID NO: 5),        and VL-CDR3 of LQSQTYPLT (SEQ ID NO: 6);    -   2) antibody ZIL1: variable heavy (VH)-CDR1 of SYGMS (SEQ ID NO:        13), VH-CDR2 of HINSGGSSTYYADAVKG (SEQ ID NO:14), VH-CDR3 of        VYTTLAAFWTDNFDY (SEQ ID NO: 15), variable light (VL)-CDR1 of        SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 of SDGNRPS (SEQ ID NO:        17, and VL-CDR3 of QSFDTTLDAYV (SEQ ID NO:18);    -   3) antibody ZIL8: VH-CDR1 of DYAMS (SEQ ID NO: 19), VH-CDR2 of        GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 of GFPGSFEH (SEQ ID        NO: 21), VL-CDR1 of TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 of        YNSDRPS (SEQ ID NO: 23), VL-CDR3 of SVYDRTFNAV (SEQ ID NO: 24);    -   4) antibody ZIL9: VH-CDR1 of SYDMT (SEQ ID NO: 25), VH-CDR2 of        DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 of LGVRDGLSV (SEQ ID        NO: 27), VL-CDR1 of SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 of        RDTERPS (SEQ ID NO: 29), VL-CDR3 of ESAVDTGTLV (SEQ ID NO: 30);    -   5) antibody ZIL11: VH-CDR1 of TYVMN (SEQ ID NO: 31), VH-CDR2 of        SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 of SMVGPFDY (SEQ ID        NO: 33), VL-CDR1 of SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 of        KDTERPS (SEQ ID NO: 35), VL-CDR3 of ESAVSSDTIV (SEQ ID NO: 36);    -   6) antibody ZIL69: VH-CDR1 of SYAMK (SEQ ID NO: 37), VH-CDR2 of        TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 of GNAESGCTGDHCPPY        (SEQ ID NO: 39), VL-CDR1 of SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2        of KDTERPS (SEQ ID NO: 41), VL-CDR3 of ESAVSSETNV (SEQ ID NO:        42);    -   7) antibody ZIL94: VH-CDR1 of TYFMS (SEQ ID NO: 43), VH-CDR2 of        LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 of FWRAFND (SEQ ID        NO: 45), VL-CDR1 of GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 of        DTGSRPS (SEQ ID NO: 47), VL-CDR3 of SLYTDSDILV (SEQ ID NO: 48);    -   8) antibody ZIL154: VH-CDR1 of DRGMS (SEQ ID NO: 49), VH-CDR2 of        YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 of WDGSSFDY (SEQ ID        NO: 51), VL-CDR1 of KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 of        KVSNRDP (SEQ ID NO: 53), VL-CDR3 of MQAIHFPLT (SEQ ID NO: 54);    -   9) antibody ZIL159: VH-CDR1 of SYVMT (SEQ ID NO: 55), VH-CDR2 of        GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 of GDIVATGTSY (SEQ ID        NO: 57), VL-CDR1 of SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 of        KDTERPS (SEQ ID NO: 59), VL-CDR3 of KSAVSIDVGV (SEQ ID NO: 60);    -   10) antibody ZIL171: VH-CDR1 of TYVMN (SEQ ID NO: 61), VH-CDR2        of SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 of SMVGPFDY (SEQ        ID NO: 63), VL-CDR1 of SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 of        KDTERPS (SEQ ID NO: 65), VL-CDR3 of ESAVSSDTIV (SEQ ID NO: 66);        or    -   11) a variant of 1) to 10) that differs from respective parent        antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154,        ZIL159, or ZIL171 by addition, deletion, and/or substitution of        one or more amino acid residues in at least one of VH or VL        CDR1, CDR2, or CDR3.

In some embodiments, the mimotope employed in the diagnostic methods ofthe present invention binds to an anti-IL-31 antibody or antigen-bindingportion thereof which binds to feline IL-31, wherein the antibodyincludes a VL chain comprising Framework 2 (FW2) changes selected fromthe following: an Asparagine in place of Lysine at position 42, anIsoleucine in place of Valine at position 43, a Valine in place ofLeucine at position 46, an Asparagine in place of Lysine at position 49,and combinations thereof, wherein the positions are in reference to thenumbering of SEQ ID NO: 127 (FEL_15H05_VL1).

Anti-IL-31 antibodies, polypeptides, and/or peptides of the presentinvention, and IL-31 peptide mimotopes are useful for immunoassays whichdetect or quantitate IL-31, or anti-IL-31 antibodies, in a sample. Animmunoassay for IL-31 typically comprises incubating a clinical orbiological sample in the presence of a detectably labeled high affinity(or high avidity) anti-IL-31 antibody, polypeptide, or peptide of thepresent invention capable of selectively binding to IL-31, and detectingthe labeled polypeptide, peptide or antibody which is bound in a sample.In a preferred embodiment, an IL-31 mimotope is bound to a solid surfaceand is used to capture a labeled anti-IL-31 antibody, such that alabeled anti-IL-31 antibody:IL-31 mimotope complex becomes tethered tothe solid surface. The labeled anti-IL-31 antibody in the complex has anaffinity to the mimotope in the complex that is lower than its affinityto the IL-31 in the sample.

The level of IL-31 in the sample can therefore be determined bymeasuring the signal coming from labeled antibody which is liberatedfrom the solid surface when the IL-31 in the sample binding to thelabeled anti-IL-31 antibody of the anti-IL-31 antibody:IL-31 mimotopecomplex. In this instance, the level of IL-31 in the sample is inverselyproportional to the signal. Various clinical assay procedures are wellknown in the art. See, e.g., IMMUNOASSAYS FOR THE 80'S (Voller et al.,eds., Univ. Park, 1981). Such samples include tissue biopsy, blood,serum, and fecal samples, or liquids collected from animal subjects andsubjected to ELISA analysis as described below.

In some embodiments, the binding of antigen to antibody is detectedwithout the use of a solid support. For example, the binding of antigento antibody can be detected in a liquid format.

In other embodiments, an IL-31 peptide mimotope, or an anti-IL-31antibody, polypeptide, or peptide can, for example, be fixed tonitrocellulose, or another solid support which is capable ofimmobilizing cells, cell particles or soluble proteins. The support canthen be washed with suitable buffers followed by treatment with thedetectably labeled IL-31-specific polypeptide, peptide or antibody. Thesolid phase support can then be washed with the buffer a second time toremove unbound polypeptide, peptide or antibody. The amount of boundlabel on the solid support can then be detected by known method steps.

“Solid phase support” or “carrier” refers to any support capable ofbinding polypeptide, peptide, antigen, or antibody. Well-known supportsor carriers, include glass, polystyrene, polypropylene, polyethylene,polyvinylidenefluoride (PVDF), dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor the purposes of the present invention. The support material can havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to the IL-31 peptide mimotope, IL-31 oran anti-IL-31 antibody. It is envisioned that the IL-31 mimotope boundto the support may itself be conjugated to a carrier polypeptide, ifdesired. Thus, the support configuration can be spherical, as in a bead,or cylindrical, as in the inside surface of a test tube, or the externalsurface of a rod. Alternatively, the surface can be flat, such as asheet, culture dish, test strip, etc. For example, supports may includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody, polypeptide, peptide or antigen,or can ascertain the same by routine experimentation.

Well known method steps can determine binding activity of a given lot ofmimotope or anti-IL-31 polypeptide, peptide and/or antibody. Thoseskilled in the art can determine operative and optimal assay conditionsby routine experimentation.

Detectably labeling an IL-31-specific polypeptide, peptide and/orantibody as well as labeling of an IL-31 peptide mimotope (or conjugatethereof) can be accomplished by several different methods, includinglinking to an enzyme for use in an enzyme immunoassay (EIA), orenzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts withthe exposed substrate to generate a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or by visualmeans. Enzymes which can be used to detectably label the IL-31-specificantibodies or mimotopes described herein include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.

By radioactively labeling the IL-31-specific antibodies or mimotopes, itis possible to detect IL-31 through the use of a radioimmunoassay (RIA).See Work et al., LAB. TECHNIQUES & BIOCHEM. 1N MOLEC. Bio. (No. HollandPub. Co., NY, 1978). The radioactive isotope can be detected by suchmeans as the use of a gamma counter or a scintillation counter or byautoradiography. Isotopes which are particularly useful for the purposeof the present invention include: ³H, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, and ¹²⁵I.

It is also possible to label the IL-31-specific antibodies or mimotopeswith a fluorescent compound. When the fluorescent labeled antibody isexposed to light of the proper wave length, its presence can then bedetected due to fluorescence. Among the most commonly used fluorescentlabeling compounds are fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

The IL-31-specific antibodies or mimotopes can also be detectablylabeled using fluorescence-emitting metals such a ¹²⁵Eu, or others ofthe lanthanide series. These metals can be attached to theIL-31-specific antibody or mimotope using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-tetraaceticacid (EDTA).

The IL-31-specific antibodies also can be detectably labeled by couplingto a chemiluminescent compound. The presence of the chemiluminescentlylabeled antibody or mimotope is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

Likewise, a bioluminescent compound can be used to label the mimotope,or IL-31-specific antibody, portion, fragment, polypeptide, orderivative thereof. Bioluminescence is a type of chemiluminescence foundin biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Detection of the mimotope, IL-31-specific antibody, portion, fragment,polypeptide, or derivative can be accomplished by a scintillationcounter, for example, if the detectable label is a radioactive gammaemitter, or by a fluorometer, for example, if the label is a fluorescentmaterial. In the case of an enzyme label, the detection can beaccomplished by colorometric methods which employ a substrate for theenzyme. Detection can also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

For the purposes of the present invention, the IL-31 which is detectedby the above assays can be present in a biological sample. Any samplecontaining IL-31 may be used. For example, the sample is a biologicalfluid such as, for example, blood, serum, lymph, urine, feces,inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissueextract or homogenate, and the like. The invention is not limited toassays using only these samples, however, it being possible for one ofordinary skill in the art, in light of the present specification, todetermine suitable conditions which allow the use of other samples.

In situ detection can be accomplished by removing a histologicalspecimen from an animal subject, and adding a labeled antibody (alone orin a complex with an IL-31 mimotope described herein) to such aspecimen. It is also envisioned that the antibody in the complex maycomprise only a portion of the antibody. The antibody (or portionthereof) may be provided by applying or by overlaying the labeledantibody (or portion) to a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of IL-31but also the distribution of IL-31 in the examined tissue. Using thepresent invention, those of ordinary skill will readily perceive thatany of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

The mimotope, antibody, fragment or derivative of the present inventioncan be adapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabeled antibody (or fragment of antibody) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantification of the ternary complex formed between solid-phaseantibody, antigen, and labeled antibody.

The antibodies or antibody:mimotope complexes may be used toquantitatively or qualitatively detect the IL-31 in a sample or todetect presence of cells that express the IL-31. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody or antibody:mimotope complex (see below) coupled withfluorescence microscopy, flow cytometric, or fluorometric detection. Fordiagnostic purposes, the antibodies may either be labeled or unlabeled.Unlabeled antibodies can be used in combination with other labeledantibodies (second antibodies) that are reactive with the antibody, suchas antibodies specific for canine or feline immunoglobulin constantregions. Alternatively, the antibodies can be directly labeled. A widevariety of labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands(particularly haptens), etc. Numerous types of immunoassays, such asthose discussed previously are available and are well known to thoseskilled in the art.

In one embodiment, the diagnostic method for detecting IL-31 is alateral flow immunoassay test. This is also known as theimmunochromatographic assay, Rapid ImmunoMigration (RIM™) or strip test.Lateral flow immunoassays are essentially immunoassays adapted tooperate along a single axis to suit the test strip format. A number ofvariations of the technology have been developed into commercialproducts, but they all operate according to the same basic principle. Atypical test strip consists of the following components: (1) samplepad—an absorbent pad onto which the test sample is applied; (2)conjugate or reagent pad—this contains antibodies specific to the targetanalyte conjugated to colored particles (usually colloidal goldparticles, or latex microspheres); (3) reaction membrane—typically ahydrophobic nitrocellulose or cellulose acetate membrane onto whichanti-target analyte antibodies are immobilized in a line across themembrane as a capture zone or test line (a control zone may also bepresent, containing antibodies specific for the conjugate antibodies);and (4) wick or waste reservoir—a further absorbent pad designed to drawthe sample across the reaction membrane by capillary action and collectit. The components of the strip are usually fixed to an inert backingmaterial and may be presented in a simple dipstick format or within aplastic casing with a sample port and reaction window showing thecapture and control zones.

There are two main types of lateral flow immunoassay used inmicrobiological testing: double antibody sandwich assays and competitiveassays. In the double antibody sandwich format, the sample migrates fromthe sample pad through the conjugate pad where any target analytepresent will bind to the conjugate. The sample then continues to migrateacross the membrane until it reaches the capture zone where thetarget/conjugate complex will bind to the immobilized antibodiesproducing a visible line on the membrane. The sample then migratesfurther along the strip until it reaches the control zone, where excessconjugate will bind and produce a second visible line on the membrane.This control line indicates that the sample has migrated across themembrane as intended. Two clear lines on the membrane is a positiveresult. A single line in the control zone is a negative result.Competitive assays differ from the double antibody sandwich format inthat the conjugate pad contains antibodies that are already bound to thetarget analyte, or to an analogue of it. If the target analyte ispresent in the sample it will therefore not bind with the conjugate andwill remain unlabelled. As the sample migrates along the membrane andreaches the capture zone an excess of unlabelled analyte will bind tothe immobilized antibodies and block the capture of the conjugate, sothat no visible line is produced. The unbound conjugate will then bindto the antibodies in the control zone producing a visible control line.A single control line on the membrane is a positive result. Two visiblelines in the capture and control zones is a negative result. However, ifan excess of unlabelled target analyte is not present, a weak line maybe produced in the capture zone, indicating an inconclusive result.There are a number of variations on lateral flow technology. The capturezone on the membrane may contain immobilized antigens orenzymes—depending on the target analyte—rather than antibodies. It isalso possible to apply multiple capture zones to create a multiplextest. For example, commercial test strips able to detect both EHEC Shigatoxins ST1 and ST2 separately in the same sample have been developed.

Importantly, the mimotopes and antibodies described herein may behelpful in diagnosing a pruritic and/or allergic in dogs, cats, orhorses. More specifically, the antibody in the antibody:mimotope complexmay bind to IL-31 in the sample and help identify the overexpression ofIL-31 in mammals, including companion animals. Thus, the antibodiesdescribed herein, which can be used in conjunction with the mimotope,may provide an important immunohistochemistry tool. In one embodiment,an assay design is conceived here whereby an IL-31 mimotope (peptide) isused to capture an antibody of the present invention that is labeled fordetection in an assay. This captured antibody would have an affinity tothe attached mimotope that is lower that the affinity of nativecirculating IL-31 in a host species. In this embodiment, incubation ofthe fluid derived from the host species is incubated with the labeledantibody: mimotope complex that is tethered to a solid surface. Thepresence of IL-31 in the test fluid derived from the host species willhave a higher affinity to the antibody, thus liberating the labeledantibody from the solid surface where it can be removed during washsteps. The level of IL-31 in the test fluid can thus be correlated tothe lack of signal that appears on the mimotope-bound surface. It isconceived that such an assay would have utility to measure IL-31 in aresearch or clinical setting for use as a diagnostic test.

The antibodies and mimotopes described herein may be used on arrays,highly suitable for measuring gene expression profiles.

Kits

Also included within the scope of the present invention are kits forpracticing the subject therapeutic methods and diagnostic methods. Inone embodiment, a kit according to the present invention at leastincludes a vaccine composition of the present invention. In oneembodiment, a vaccine of the present invention may be provided, usuallyin a lyophilized form, in a container. In another embodiment, a kitaccording to the present invention can include the components necessaryto carry out the diagnostic methods of this invention. For example, akit of this invention may include as one of its components an IL-31mimotope as described herein, such as a feline IL-31 mimotope, a canineIL-31 mimotope, a horse IL-31 mimotope, or a human IL-31 mimotope. Sucha mimotope may already be bound to a solid surface. A kit according tothe present invention can also include antibodies. The antibodies, whichmay be conjugated to a label or toxin, or unconjugated, are typicallyincluded in the kits with buffers, such as Tris, phosphate, carbonate,etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or thelike. Generally, these materials will be present in less than 5% wt.based on the amount of active antibody, and usually present in totalamount of at least about 0.001% wt. based again on the antibodyconcentration. Frequently, it will be desirable to include an inertextender or excipient to dilute the active ingredients, where theexcipient may be present in from about 1% to 99% wt. of the totalcomposition. Where a second antibody capable of binding to the primaryantibody is employed in an assay, this will usually be present in aseparate vial. The second antibody is typically conjugated to a labeland formulated in an analogous manner with the antibody formulationsdescribed above. The kits will generally also include a set ofinstructions for use.

In one embodiment, a kit according to the present invention is a teststrip kit (lateral flow immunoassay kit) useful for detecting IL-31,such as canine, feline, equine, or human IL-31 protein in a sample. Sucha test strip will typically include a sample pad onto which the testsample is applied; a conjugate or reagent pad containing an antibody orantibody:mimotope specific to canine, feline, equine, or human IL-31,wherein the antibody or antibody:mimotope complex is conjugated tocolored particles (usually colloidal gold particles); a reactionmembrane onto which anti-IL-31 antibodies or an antibody:mimotopecomplex are immobilized in a line across the membrane as a capture zoneor test line (a control zone may also be present, containing antibodiesor an antibody:mimotope complex specific for the conjugate antibodies);and a further absorbent pad designed to draw the sample across thereaction membrane by capillary action and collect it. The test strip kitwill generally also include directions for use.

The invention will now be described further by the non-limiting examplesbelow. In the example section below and in the figures, any datapresented for antibodies containing “11E12” in their designation is forpurposes of comparison with the antibodies of the present invention.

EXAMPLES 1. Example 1

1.1. Production of Canine Interleukin 31 (cIL-31) from Chinese HamsterOvary (CHO) Cells

The Interleukin 31 protein varies in amino acid sequence conservationamong homologous species (FIGS. 1A and 1B) but is believed to havecommon structural architecture with other members of the type I cytokinefamily (Boulay et al. 2003, Immunity. August; 19(2):159-632003; Dillonet al. 2004 Nat Immunol. July; 5(7):752-60). This up-down bundletopology is significant to the mode of receptor recognition shared bythese cytokines (Dillon et al. supra, Cornelissen et al. 2012 Eur J CellBiol. June-July; 91(6-7):552-66). With variation in IL-31 proteinsequence identities between different species, it is not possible topredict if antibodies raised against one species will cross-react withothers given different epitope propensities and local amino acidcompositions. As a consequence, multiple forms of IL-31 protein wereconsidered for this work representing multiple species and expressionsystems. The canine IL-31 protein (cIL-31) was produced to use as animmunogen and a reagent to test affinity and potency of antibody hits.Recombinant cIL-31 was created in CHO cells using the CHROMOS ACE(Artificial Chromosome Expression) system (Chromos Molecular Systems,Inc., Burnaby, British Columbia) to generate the secreted canine IL-31protein having the sequence of (SEQ ID NO: 155; Canine_IL31), thecorresponding nucleotide sequence for which is (SEQ ID NO: 156;Canine_IL31). Conditioned medium from 400 ml of cell culture (CHO cellline) was obtained and dialyzed against 10 volumes of QA buffer (20 mMTris pH 8.0, 20 mM NaCl) for 4.5 hours. Dialyzed medium was 0.2 μmfiltered and loaded at 1 ml/min onto a SOURCE™ Q column (GE Healthcare,Uppsala, Sweden) pre-equilibrated with QA buffer. Protein was elutedusing a multi-step linear gradient. The majority of cIL-31 remained inthe flow through (FT) fraction, a small amount of cIL-31 eluted early inthe gradient. Identity of the protein was previously confirmed byWestern immunoblotting, and Mass-Spectrometry (MS) analysis of a trypticdigest. Protein in the FT fraction was concentrated 4-5 fold anddialyzed overnight against Phosphate Buffered Saline (PBS) at 4° C.Stability of the protein was checked following dialysis into PBS. Noprecipitation was observed, and no proteolysis was observed afterseveral days at 4° C. De-glycosylation experiments using N-glycosidase Fresulted in the protein condensing down to a single band of ˜15 kDa onSDS-PAGE. Protein concentration was determined using a bicinchoninicassay (BCA assay) with Bovine Serum Albumin (BSA) as a standard(ThermoFisher Scientific, Inc., Rockford, Ill.). The protein solutionwas split into aliquots, snap frozen (liquid N₂) and stored at −80° C.

1.2. Transient Expression of Wildtype and Mutant Feline Interleukin 31(fIL-31) from CHO Cells

To aid in the identification of antibodies with the appropriate epitopebinding property, wildtype and mutant feline IL-31 proteins wereexpressed in a mammalian expression system for production, purification,and assessment in affinity and cell-based assays. The binding site ofantibody 11E12 on IL-31 was described previously (U.S. Pat. No.8,790,651 to Bammert, et al.). Characterization of the novel bindingsite on IL-31 recognized by antibody 15H05 is described herein. Thewildtype designation is full length feline IL-31 protein with no changesto the native amino acid residues. Mutant proteins were designated bytheir corresponding antibodies name (11E12 and 15H05) referring tomutations in amino acids in the IL-31 protein that (when altered) affectthe binding to each respective antibody. Identification of theappropriate mutations required for the feline IL-31 15H05 protein aredescribed below in section 1.10. The objective was to change amino acidsin the IL-31 epitope and observe a loss of binding phenotype to eachrespective antibody. A comparison can then be made during screening tosee if new candidate antibodies bind to the wildtype protein and not tothe mutant. New antibody hits can then be binned according to binding tothe same or similar epitope as antibody 11E12 or 15H05.

Expression constructs were codon optimized and synthesized forexpression in Chinese Hamster Ovarian (CHO) cells. The synthesized geneswere cloned into pD2529 (ATUM vector) for transient expression use.Wildtype feline IL-31 protein is represented by (SEQ ID NO: 157;Feline_IL31_wildtype), the corresponding nucleotide sequence for whichis (SEQ ID NO: 158; Feline_IL31_wildtype). Mutant feline IL-31 11E12protein is represented by (SEQ ID NO: 161; Feline_IL31_11E12_mutant),the corresponding nucleotide sequence for which is (SEQ ID NO: 162;Feline_IL31_11E12_mutant). Mutant feline IL-31 15H05 protein isrepresented by (SEQ ID NO: 163; Feline_IL31_15H05_mutant), thecorresponding nucleotide sequence for which is (SEQ ID NO: 164;Feline_IL31_15H05_mutant). Recombinant feline IL-31 proteins wereexpressed in ExpiCHO-S™ cells (ThermoFisher Scientific, Inc., Rockford,Ill.) by following the manufacturer's maximum titer protocol fortransient CHO expression. Twelve days post transfection the cells werecentrifuged and filtered to capture the secreted protein in theconditioned media. For each construct (wild type and mutants), 120 mL ofconditioned media (from CHO cell culture, 0.2 μm filtered) was adjustedto 30 mS/cm with the addition of NaCl, 5 mM imidazole, and pH 7.4. Eachmedia sample was combined with 5 mL of HisPur Cobalt resin (ThermoFisherScientific, Inc., Rockford, Ill.) which had been equilibrated with 5 mMimidazole, 20 mM sodium phosphate, 300 mM NaCl, pH 7.4. Each sample andresin was allowed to mix at 4° C., overnight. Resins were collected (andseparated from the unbound fraction) by pouring through BioRadEconcolumns (Bio-Rad, Hercules, Calif.). Resins were washed with 5×5 mLof buffer (as above) and then eluted with 5×5 mL of 500 mM imidazole inthe same buffer. Fractions were evaluated by SDS-PAGE. Concentration wasmeasured by a BCA protein assay using standard methods.

1.3. Production of Feline Interleukin 31 (fIL-31) from E. Coli

Recombinant feline IL-31 protein was generated in an E. coli expressionhost to use as an assay reagent and for in vivo challenge studies toinduce a pruritic response in cats. The gene representing feline IL-31was synthesized for optimal expression in E. coli. Expression constructswere created with the full-length feline IL-31 gene containing anN-terminal 6-His tag for detection and purification. This feline IL-31protein is represented by (SEQ ID NO: 159; Feline_IL-31_E_coli), thecorresponding nucleotide sequence for which is (SEQ ID NO: 160;Feline_IL-31_E_coli). Sequence confirmed plasmids were used to transformE. coli BL21 DE3 (Invitrogen Corp., Carlsbad, Calif.) and subsequentprotein expression carried out.

Cell paste (262.3 g) from E. coli was lysed as follows: The cell pastewas resuspended in 500 mL of 50 mM Tris pH 8, filtered through astainless mesh filter to remove particles, and then lysed via two passesthrough a microfluidizer at 1300 psi. The lysate (1200 mL volume) wasdivided into four bottles, centrifuged at 12,000 g for 20 minutes at 10°C. The supernatant was decanted and discarded. Each pellet was washed bysuspension in 300 mL of 5 mM EDTA, 0.5% Triton X-100, pH 9.0, and thencentrifugation at 12,000 g, 50 minutes, at 10° C. The supernatant wasdecanted and discarded. The washed pellets were stored at −20° C. untilfolding and isolation.

Prior to isolation, one of the pellets was washed with water to removethe residual detergent, and then centrifuged at 10,000 g, 20 minutes, at4° C. Again, the supernatant was decanted. Finally, the washed pelletwas solubilized in 60 mL of 50 mM sodium phosphate, 300 mM NaCl, 6 Mguanidine-HCl, 5 mM imidazole, pH 7.4. The pellet was allowed to mix atroom temperature for approximately 25 minutes before centrifuging againat 10,000 g, 20 minutes, and 4° C. This time, the supernatant wasdecanted and kept for further processing. The pellet (resuspended inwater to original volume) was set aside for SDS-PAGE only. Crude IMAC(immobilized metal affinity chromatography) was performed to increasepurity prior to folding. In this case, 15 mL of Ni-NTA Superflow (QiagenInc, Germantown, Md., Cat #30450, pre-equilibrated in the same buffer)was added to the clarified supernatant and allowed to mix at roomtemperature for approximately 90 minutes. The unbound fraction wasdecanted and set aside for SDS-PAGE. The IMAC resin was washed with 5 mMimidazole, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, pH7.4 (same as solubilization buffer). The resin was eluted with (first7.5 mL and then multiples of 15 mL, monitoring protein elution byBradford assay) 200 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 6M guanidine-HCl, pH 7.4. Elution fractions containing protein (as perBradford) were pooled (125 mL) for further processing.

The IL-31 protein was folded as follows. The IL-31 was reduced by theaddition of dithiothreitol to a final of 10 mM, and allowed to mix atroom temperature for 2 hours. The diluted sample was then diluteddrop-wise into 2500 mL (20× volume) of PBS+1 M NaCl with rapid stirring.The theoretical concentration of urea should have been approximately 0.4M at this point. The remainder of the urea was removed slowly bydialysis against 3 exchanges (4 L each) of PBS, at 4° C. overnight.Following dialysis, the sample was 0.2 μm filtered to remove anyunfolded/precipitated protein.

The sample was further purified by a second round of IMAC, this timewith a linear gradient elution. Fifteen mL of Ni-NTA Superflow resin wasadded to the sample and allowed to bind batch-wise by stirring (with asuspended stir bar) overnight at 4° C. Again, the unbound fraction wasdecanted and set aside. The Ni-NTA Superflow resin was packed in an XK16column (GE Healthcare Lifesciences, Marlborough, Mass.) and hooked up toan AKTA brand chromatography system (GE Healthcare Lifesciences,Marlborough, Mass.). The column was then washed with 50 mM Tris, 300 mMNaCl, pH 8.2 and the eluted via a 150 mL linear gradient from 0 to 500mM imidazole, each in wash buffer. Fractions were analyzed by SDS-PAGE.Fractions having sufficient purity of IL-31 were pooled and bufferexchanged again by dialysis against 3 exchanges (2 L each) of PBS, at 4°C., overnight. Finally, the folded and purified sample was collectedfrom dialysis, sterile filtered, concentration measured aliquoted,snap-froze in a dry-ice/isopropanol bath, and stored at −80° C.

1.4. Method to Determine Affinity of Anti-IL-31 Antibodies for IL-31Using Surface Plasmon Resonance

The affinity with which candidate mAbs bind canine and feline IL-31 wasdetermined using surface plasmon resonance (SPR) on a Biacore system(Biocore Life Sciences (GE Healthcare), Uppsala, Sweden). To avoidaffinity differences associated with differential surface preparationthat can occur when immobilizing antibodies to surfaces; a strategy wasemployed where IL-31 was directly conjugated to the surface.Immobilization was obtained by amine coupling 5 μg/mL IL-31 usingN-hydroxysuccinimide (NHS)/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. Chips were quenched with ethanolamine andthe affinity with which all candidate mAbs bound to the immobilizedIL-31 was evaluated. All curves were fit to a 1:1 model. Affinityconstants (KD) less than 1×10⁻¹¹ M (1E-11 M) are below the lower limitof quantitation of detection for the instrument. Results for affinitymeasurements are described herein.

1.5. Method to Determine Potency of Anti-IL-31 Antibodies Assessed byInhibition of Canine and Feline IL-31 Induced pSTAT3 Signaling in Canineand Feline Macrophage Cells

To identify candidates with inhibitory activity, antibodies wereassessed for their ability to affect IL-31-mediated STAT3phosphorylation in either a canine or feline cell-based assay. STAT3phosphorylation was determined in canine DH-82 (ATCC® CRL-10389™) orfeline FCWF4 macrophage-like cells (ATCC CRL-2787). DH82 and FCWF4 cellswere primed with canine interferon gamma (R&D Systems, Minneapolis,Minn.) at 10 ng/mL for 24 hours or feline interferon gamma (R&D Systems,Minneapolis, Minn.) at 125 ng/mL for 96 hours, respectively, to increasereceptor expression. Both cell types were serum starved for 2 hoursprior to IL-31 and mAb treatment. Using two independent methods, allcandidate mAbs were evaluated for their ability to inhibit either 1μg/mL canine or 0.2 μg/mL feline IL-31 induced STAT3 phosphorylation.Assays were also run to demonstrate cross-reactivity of canine andfeline cytokines and cross-functionality of the antibodies ability toinhibit signaling in both species. To ensure complex formation, a onehour co-incubation of mAb and IL-31 cytokine prior to cell stimulationwas completed. IL-31 cell stimulation was carried out for five minutes.STAT3 phosphorylation was measured using AlphaLISA SureFire ULTRA™technology (Perkin Elmer, Waltham, Mass.). In the case where antibodyconcentration and purity are unknown, hybridoma supernatants werequalitatively measured for their ability to inhibit STAT3phosphorylation following a 1 hour co-incubation with 1 mg/ml canine or0.2 mg/ml feline IL-31. The potency of individual monoclonal antibodiesdefined by their ability to inhibit IL-31 mediated STAT3 phosphorylationin these assays was considered the key selection criteria for furtheradvancement of select antibodies. The term potency refers to the IC50value calculated from these assays and is the concentration of theantibody where signaling induced by IL-31 is reduced to one half itsmaximal value. Increased potency described herein correlates to a lowerIC50 value.

1.6. Identification of Mouse and Canine Monoclonal AntibodiesRecognizing Canine and Feline Interleukin 31

Mice and dogs were immunized with recombinant canine IL-31 (SEQ ID No.155) for the purpose of identifying antibodies. Serum antibody titersfrom immunized animals were determined using an enzyme linkedimmunosorbent assay (ELISA). Canine or feline IL-31 (50 ng/well) wasimmobilized to polystyrene microplates and used as a capture antigen.Serum from immunized animals was diluted in phosphate buffered salinewith 0.05% tween-20 (PBST). The presence of anti-IL-31 antibodies wasdetected with an appropriate secondary HRP labeled antibody. Followingaddition of a chromogenic substrate (SureBlue Reserve TMB 1-ComponentMicrowell Peroxidase Substrate, KPL, Inc., Gaithersburg, Md.) and a tenminute incubation at room temperature (RT) the reaction was stopped withthe addition of 100 μL of 0.1 N HCl. The absorbance of each well wasdetermined at an optical density (OD) of 450 nm. Antibodies wereselected for their ability to bind canine and feline IL-31 using anELISA. In some cases, further characterization was performed at the timeof selection using an ELISA with a mutant form of the feline IL-31protein as a capture antigen. Cells producing antibodies with desiredbinding and inhibitory properties were chosen for sequence analysis ofRNA transcripts representing the variable heavy (VH) and variable light(VL) IgG chains.

In the case of mouse antibodies, donor splenocytes from a singleresponsive CF-1 mouse were used for fusion and hybridoma supernatantswere screened for antibodies that bind to either canine or feline IL-31proteins by ELISA. This resulted in the identification of a single mouseantibody, Mu-15H05, having a sub-nanomolar affinity to both species ofIL-31 (FIG. 2, section A). Mouse anti IL-31 15H05 was further subclonedto generate a hybridoma producing homogeneous antibody and forsequencing of the variable heavy and light chains. The mouse anti IL-31variable sequences determined for antibody 15H05 are as follows, 15H05variable heavy chain (SEQ ID NO: 67; MU-15H05-VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 68; MU-15H05-VH), 15H05variable light chain (SEQ ID NO: 69; MU-15H05-VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 70; MU-15H05-VL). Inaddition to mouse antibody 15H05, further consideration was given tomouse-derived antibody 11E12 that was previously described in U.S. Pat.No. 8,790,651 to Bammert, et al. Described herein are data showing theability of antibody 11E12 to bind both canine and feline IL-31 proteinswith high affinity. The ability of 11E12 to bind feline IL-31 made thisantibody a suitable candidate for felinization and potential therapeuticuse in cats. The mouse anti IL-31 variable sequences previouslydetermined for antibody 11E12 are as follows, 11E12 variable heavy chain(SEQ ID NO: 71; MU-11E12-VH), the corresponding nucleotide sequence forwhich is (SEQ ID NO: 72; MU-11E12-VH), 11E12 variable light chain (SEQID NO: 73; MU-11E12-VL), the corresponding nucleotide sequence for whichis (SEQ ID NO: 74; MU-11E12-VL).

Dogs having elevated anti IL-31 titers following vaccination wereselected for analysis of B-cell populations producing antibodies withdesired phenotypes. B-cells were derived from PBMCs, bone marrow,spleen, or lymph nodes for further analysis. Single B-cells weresegregated into individual wells and assayed for presence of secretedIgGs capable of binding wildtype, 11E12 mutant, and 15H05 mutant formsof canine IL-31 (AbCellera, Vancouver, BC) using methods described inUS2012/0009671A1, US2016/0252495A1, U.S. Pat. No. 9,188,593, WO2015/176162 A9, and WO 2016/123692 A1.

This screening strategy is based on known regions of the IL-31 proteinthat are critical for binding and signal transduction through itsco-receptor complex. Selection of these mutant proteins for screening isdescribed in section 1.2 of this application. Sequencing of the variableheavy and light IgG domains was carried following an RT-PCR reactionfrom individual candidate B-cells. These screens resulted in theidentification of nine canine antibodies selected for furtherevaluation. These canine anti IL-31 variable sequences are as follows,ZIL1 variable heavy chain (SEQ ID NO:75; CAN-ZIL1_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 76; CAN-ZIL1_VH), ZIL1variable light chain (SEQ ID NO: 77; CAN-ZIL1_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 78; CAN-ZIL1_VL); ZIL8variable heavy chain (SEQ ID NO:79; CAN-ZIL8_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 80; CAN-ZIL8_VH), ZIL8variable light chain (SEQ ID NO: 81; CAN-ZIL8_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 82; CAN-ZIL8_VL); ZIL9variable heavy chain (SEQ ID NO:83; CAN-ZIL9_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 84; CAN-ZIL9_VH), ZIL9variable light chain (SEQ ID NO: 85; CAN-ZIL9_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 86; CAN-ZIL9_VL); ZIL11variable heavy chain (SEQ ID NO:87; CAN-ZIL11_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 88; CAN-ZIL11_VH), ZIL11variable light chain (SEQ ID NO: 89; CAN-ZIL11_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 90; CAN-ZIL11_VL); ZIL69variable heavy chain (SEQ ID NO:91; CAN-ZIL69_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 92; CAN-ZIL69_VH), ZIL69variable light chain (SEQ ID NO: 93; CAN-ZIL69_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 94; CAN-ZIL69_VL); ZIL94variable heavy chain (SEQ ID NO:95; CAN-ZIL94_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 96; CAN-ZIL94_VH), ZIL94variable light chain (SEQ ID NO: 97; CAN-ZIL94_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 98; CAN-ZIL94_VL); ZIL154variable heavy chain (SEQ ID NO:99; CAN-ZIL154_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 100; CAN-ZIL154_VH), ZIL154variable light chain (SEQ ID NO: 101; CAN-ZIL154_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 102; CAN-ZIL154_VL); ZIL159variable heavy chain (SEQ ID NO:103; CAN-ZIL159_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 104; CAN-ZIL159_VH), ZIL159variable light chain (SEQ ID NO: 105; CAN-ZIL159_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 106; CAN-ZIL159_VL); ZIL171variable heavy chain (SEQ ID NO:107; CAN-ZIL171_VH), the correspondingnucleotide sequence for which is (SEQ ID NO: 108; CAN-ZIL171_VH), ZIL171variable light chain (SEQ ID NO: 109; CAN-ZIL171_VL), the correspondingnucleotide sequence for which is (SEQ ID NO: 110; CAN-ZIL171_VL).

The aforementioned nine monoclonal antibodies which were selected forfurther characterization may be referred to elsewhere in thespecification, figures, or claims as ZIL1, ZIL8, ZIL8, ZIL11, ZIL69,ZIL94, ZIL154, ZIL159, and ZIL171.

1.7. Construction of Recombinant Chimeric and Fully Canine Antibodies

Antibody variable domains are responsible for antigen binding. Graftingof the full variable domain onto respective constant region is expectedto have little or no impact on the antibody's ability to bind the IL-31immunogen. To simultaneously confirm that the correct sequences of theheavy and light chain variable regions were identified and to producehomogenous material, expression vectors were designed to producerecombinant chimeric or fully canine antibodies in mammalian expressionsystems. Chimeric antibodies described here consist of the variablesequence (both CDR and framework) from the host species antibody graftedonto the respective heavy and light constant regions of a feline orcanine IgG molecule (for example; mouse variable: canine constant isreferred to as mouse:canine chimera). Fully canine antibodies describedhere consist of the variable sequence (both CDR and framework) from thehost species antibody (canine) grafted on to the respective heavy andlight chain constant regions of the canine IgG molecule. Synthetic DNAsequences were constructed for the variable heavy (VH) and variablelight (VL) sequences of selected antibodies. These sequences containunique restriction endonuclease sites, a Kozak consensus sequence and,an N-terminal secretion leader to facilitate expression and secretion ofthe recombinant antibody from a mammalian cell line.

For mouse: feline chimeras, each respective variable region was clonedinto a mammalian expression plasmid containing either the feline IgGheavy (SEQ ID NO: 173; Feline_HC_AlleleA_1) the corresponding nucleotidesequence for which is (SEQ ID NO: 174; Feline_HC_AlleleA_1) or lightchain (SEQ ID NO: 175; Feline_LC_Kappa_G_minus) the correspondingnucleotide sequence for which is (SEQ ID NO: 176;Feline_LC_Kappa_G_minus) constant regions. For mouse: canine chimeras orfully canine antibodies, each mouse or canine variable region was clonedinto a mammalian expression plasmid containing either the canine IgGheavy (SEQ ID NO: 177; Canine_HC_65_1) the corresponding nucleotidesequence for which is (SEQ ID NO: 178; Canine_HC_65_1) or light chain(SEQ ID NO: 179; Canine_LC_Kappa) the corresponding nucleotide sequencefor which is (SEQ ID NO: 180; Canine_LC_Kappa) constant regions. Theplasmids encoding each heavy and light chain, under the control of theCMV promoter, were co-transfected into HEK 293 cells using standardmethods. Following six days of expression, chimeric mAbs were purifiedfrom 50 ml of transiently transfected HEK293FS cell supernatants usingMabSelect Sure protein A resin (GE Healthcare, Uppsala, Sweden)according to standard methods for protein purification. Eluted fractionswere pooled, concentrated to ˜500 μl using a 10,000 nominal MW cutoffNanosep Omega centrifugal device (Pall Corp., Port Washington, N.Y.),dialyzed overnight at 4° C. in 1× PBS, pH7.2 and stored at 4° C. forfurther use. Affinity and cell-based potency of select recombinantantibodies are described below.

FIG. 2 details the affinity of antibodies with CDRs derived from mouseorigin using biacore. FIG. 2, section A shows the affinity of mouse antiIL-31 antibodies 11E12 and 15H05 and the corresponding affinities of thefeline and canine chimeric forms to both feline and canine IL-31surfaces. These observations confirm the correct sequence for both mouseantibodies and indicate conversion to the chimeric form results inantibodies with equivalent or higher affinity when compared to the mouseparent with the exception of the mouse:feline 15H05 chimera which lostsome affinity to both IL-31 species as a result of its conversion to thechimeric form. Fully mouse and chimeric forms of antibodies 11E12 and15H05 were also tested for activity in the canine and feline cellularassays described in section 1.5. FIG. 3 shows the results for theseassays. Mouse antibodies 11E12 and 15H05 were tested for activityagainst canine and feline cell types using both canine and feline IL-31to stimulate signaling. The potency of both mouse antibodies wascomparable against both canine and feline cells using the felinecytokine with the exception of 15H05 against feline IL-31 in felineFCWF4 cells that shows a slight increase in IC50. Mouse 15H05 wascapable of blocking canine IL-31 signaling in both feline and caninecells with the potency in the canine assay being slightly higher. Theseresults indicate that the respective epitopes recognized by theseantibodies exists on both canine and feline IL-31 and binding of theseantibodies is capable of neutralizing receptor-mediated cellularsignaling in a relevant cell line from both species.

FIG. 3 also describes the potency of select chimeras in both cellularassays. Conversion of mouse antibodies to feline and canine chimeras hadminimal impact on the potency against feline IL-31 in the feline potencyassay (IC50 range 1.15-3.45 μg/ml). Similar results were observed whenthese chimeras were tested against feline IL-31 signaling on the canineDH82 cell line with a slight increase in potency (IC50=0.71 μg/ml)observed for the 15H05 mouse:

canine chimera. In general, there was an increase in IC50 values againstcanine IL-31 in both canine and feline cell types. The mouse:feline15H05 chimera was slightly less potent in this assay format compared tothe mouse:canine form (IC50 28.61 vs. 12.49 μg/ml). Consistent withobservations for the mouse antibodies, conversion to canine and felinechimeric forms resulted in minimal changes in potency.

Antibodies described above that were identified from single B cells ofimmunized dogs were constructed as recombinant IgG proteins followingidentification of their variable domain sequences. Grafting of thesevariable domains onto the canine heavy chain Fc (65_1 isotype) resultedin the generation of recombinant fully canine antibodies. It was ofinterest to identify additional canine antibodies that bound wildtypefeline IL-31 and who's binding was decreased to the feline IL-31 15H05mutant (i.e., are directed at the 15H05 epitope). These antibodiesobtained from this alternate source (canine vs. mouse) provideadditional paratopes (the portion of the antibody which recognizes theIL-31 protein, includes CDRs) recognizing the 15H05 epitope thusincreasing the diversity of antibodies with different physicalproperties to select from.

FIG. 4 shows the results obtained for binding of these recombinantcanine antibodies to various proteins using both ELISA and Biacoremethods. For the indirect ELISA method, antibody binding to wildtype andfeline IL-31 15H05 mutant proteins was assessed. All nine caninemonoclonal antibodies (ZIL1, ZIL8, ZIL8, ZIL11, ZIL69, ZIL94, ZIL154,ZIL159, and ZIL171) were capable of binding to wildtype feline IL-31 andbinding was impacted by mutations in the mAb 15H05 epitope regionconfirming the correct binding phenotype determined during the initialscreening used to identify them. In comparison, the 11E12 antibody boundto the wild-type feline IL-31 and its binding was not impacted by themutations in the 15H05 epitope region as evidenced by the data in FIG.4. To confirm binding, biacore analysis was performed using canine,feline, equine, human, feline 15H05 mutant, and feline 11E12 mutantIL-31 proteins as surfaces and a single test concentration of antibody.Similar to ELISA observations, all antibodies tested bound to wildtypefeline IL-31. In agreement with the data described above in thissection, mouse antibodies 11E12 and 15H05 both bound to canine andfeline IL-31 surfaces. Three additional antibodies were shown to havethis dual binding property, ZIL69 (partial canine binding), ZIL94, andZIL159. From this group of nine fully canine antibodies, only ZIL1 andZIL9 cross-reacted with equine IL-31. Of note, antibody 15H05 was theonly one of all assayed herein that bound to canine, feline, and equineIL-31 indicating some level of epitope conservation across the threespecies. In contrast, none of the antibodies described herein bound tohuman IL-31. Additional biacore surfaces were used to verify ELISAobservations showing differential binding of antibodies to wildtypefeline IL-31 and two proteins with mutations in the 15H05 (15H05 mutant)or 11E12 (11E12 mutant) epitopes. As expected, control mouse antibody11E12 bound to the 15H05 IL-31 mutant and did not bind to the 11E12IL-31 mutant due to mutations in the epitope. Likewise, mouse 15H05 didnot bind to the 15H05 mutant and retained binding to the 11E12 IL-31mutant further distinguishing the separate binding epitopes recognizedby these two antibodies. In agreement with the ELISA results, all fullycanine antibodies were impacted by the 15H05 mutation with the exceptionof ZIL94, ZIL154, and ZIL171 (partially affected). Differing results canbe attributed to differences in the two assay methodologies. Inaddition, binding of three antibodies was also shown to be impacted bythe 11E12 mutation; ZIL1 (partially effected), ZIL8, and ZIL159. Theseresults indicate the epitope recognized by these antibodies is impactedby changes in both regions of the IL-31 protein. Taken together theseresults support the characterization nine antibodies derived from canineB cells sharing binding to a region on the feline IL-31 protein that isrecognized by antibody 15H05.

1.8. Felinization of the Murine 11E12 and 15H05 Antibodies andOptimization of Binding Affinities

The generation of anti-drug antibodies (ADAs) can been associated withloss of efficacy for any biotherapeutic protein including monoclonalantibodies. Comprehensive evaluation of the literature has shown thatspeciation of monoclonal antibodies can reduce the propensity for mAbsto be immunogenic although examples of immunogenic fully human mAbs andnon-immunogenic chimeric mAbs can be found. To help mitigate risksassociated with ADA formation for the anti-IL-31 monoclonal antibodiesprovided herein, a felinization strategy was employed. This felinizationstrategy is based on identifying the most appropriate feline germlineantibody sequence for CDR grafting. Following extensive analysis of allavailable feline germline sequences for both the variable heavy andlight chain, germline candidates were selected based on their homologyto the mouse mAbs, and the CDRs from the mouse progenitor mAbs were usedto replace native feline CDRs. The objective was to retain high affinityand cell-based activity using feline antibody frameworks to minimize thepotential of immunogenicity in vivo. Felinized mAbs were expressed andcharacterized for their affinity to feline IL-31 and their potency incell-based assays. In the event that a felinized antibody loses itsability to bind IL-31, a systematic dissection was undertaken toidentify; 1) the chain responsible for the loss of function, 2) theframework responsible for the loss of function and 3) the amino acid(s)responsible for the loss function.

Synthetic nucleotide constructs representing the felinized variableheavy and light chains of mAbs 11E12 and 15H05 were made. Followingsubcloning of each variable chain into plasmids containing therespective feline heavy or kappa constant region, plasmids wereco-transfected for antibody expression in HEK 293 cells. Initialattempts at felinization of antibody 11E12 focused on utilization of asingle feline VH framework (SEQ ID NO: 111; FEL_11E12_VH1) thecorresponding nucleotide sequence for which is (SEQ ID NO: 112;FEL_11E12_VH1) paired independently with VL frameworks (SEQ ID NO: 113;FEL_11E12_VL1) the corresponding nucleotide sequence for which is (SEQID NO: 114; FEL_11E12_VL1) and (SEQ ID NO: 115; FEL_11E12_VL2) thecorresponding nucleotide sequence for which is (SEQ ID NO: 116;FEL_11E12_VL2) to form Feline 11E12 1.1 and Feline 11E12 1.2respectively. This attempt at speciation resulted in a loss of affinitywith Feline 11E12 1.1 to both the feline and canine IL-31 proteins and atotal loss of binding with the Feline 11E12 1.2 mAb when compared to themouse form of the antibody (FIG. 2, section B). Potency of thesespeciated antibodies was tested in the canine DH82 and Feline FCWF4 cellassays using the feline IL-31 cytokine. Felinized 11E12 1.1 hadapproximately a two-fold decrease in potency against feline IL-31 in thefeline FCWF assay when compared to the mouse version of the antibody. Inagreement with the loss of affinity for felinized 11E12 1.2, a completeloss of cellular potency was observed for this antibody (FIG. 3). Basedon previous experience during caninization of the mAb 11E12 ortholog, asimilar strategy was undertaken in attempt to restore the affinity lossto felinization (U.S. Pat. No. 8,790,651 to Bammert, et al.).Substitution of the felinized framework 2 (FW2) region of Feline 11E12VL1 with the mouse FW2 from (SEQ ID NO: 73; Mu_11E12_VL) thecorresponding nucleotide sequence for which is (SEQ ID NO: 74;Mu_11E12_VL) was done to generate Feline 11E12 VL1 FW2. In addition, asingle substitution at position 46 of the feline VL (K46Q) was performedto generate (SEQ ID NO: 119; FEL_11E12_VL1_K46Q) the correspondingnucleotide sequence for which is (SEQ ID NO: 120; FEL_11E12_VL1_K46Q).Pairing of the above VLs with Fel_11E12_VH1 resulted in Feline 11E12 1.1FW2 and Feline 11E12 1.1 K46Q respectively. Changing FW2 resulted in arestoration of affinity for Feline 11E12 1.1 FW2 to the feline IL-31protein resulting in a KD equivalent to that of the mouse and chimericform (FIG. 2, sections A and B). These changes however had a detrimentaleffect on Feline 11 E12 1.1 FW2s affinity to the canine IL-31 proteinindicating a clear distinction in the nature of antibody 11E12s abilityto bind this epitope on the feline and canine cytokine. The single aminoacid substitution in Feline 11E12 1.1 K46Q was unable to influenceaffinity of this antibody. Increased affinity of antibody 11E12 1.1 FW2for the feline IL-31 protein resulted in increased potency against thefeline cytokine in the canine DH82 assay (FIG. 3).

Felinization efforts with mouse antibody 15H05 focused on thecombinations of three feline VH frameworks with three feline VLframeworks for a total of 9 felinized mAbs. FEL_15H05_VH1 (SEQ ID NO:121; FEL_15H05_VH1) the corresponding nucleotide sequence for which is(SEQ ID NO: 122; FEL_15H05_VH1) was combined with (SEQ ID NO: 127;FEL_15H05_VL1) the corresponding nucleotide sequence for which is (SEQID NO: 128; FEL_15H05_VL1), (SEQ ID NO: 129; FEL_15H05_VL2) thecorresponding nucleotide sequence for which is (SEQ ID NO: 130;FEL_15H05_VL2), and (SEQ ID NO: 131; FEL_15H05_VL3) the correspondingnucleotide sequence for which is (SEQ ID NO: 132; FEL_15H05_VL3) tocreate Feline 15H05 1.1, Feline 15H05 1.2, and Feline 15H05 1.3respectively. FEL 15H05 VH2 (SEQ ID NO: 123; FEL_15H05_VH2) thecorresponding nucleotide sequence for which is (SEQ ID NO: 124;FEL_15H05_VH2) was combined with (SEQ ID NO: 127; FEL_15H05_VL1) thecorresponding nucleotide sequence for which is (SEQ ID NO: 128;FEL_15H05_VL1), (SEQ ID NO: 129; FEL_15H05_VL2) the correspondingnucleotide sequence for which is (SEQ ID NO: 130; FEL_15H05_VL2), and(SEQ ID NO: 131; FEL_15H05_VL3) the corresponding nucleotide sequencefor which is (SEQ ID NO: 132; FEL_15H05_VL3) to create Feline 15H05 2.1,Feline 15H05 2.2, and Feline 15H05 2.3 respectively. FEL_15H05_VH3 (SEQID NO: 125; FEL_15H05_VH3) the corresponding nucleotide sequence forwhich is (SEQ ID NO: 126; FEL_15H05_VH3) was combined with (SEQ ID NO:127; FEL_15H05_VL1) the corresponding nucleotide sequence for which is(SEQ ID NO: 128; FEL_15H05_VL1), (SEQ ID NO: 129; FEL_15H05_VL2) thecorresponding nucleotide sequence for which is (SEQ ID NO: 130;FEL_15H05_VL2), and (SEQ ID NO: 131; FEL_15H05_VL3) the correspondingnucleotide sequence for which is (SEQ ID NO: 132; FEL_15H05_VL3) tocreate Feline 15H05 3.1, Feline 15H05 3.2, and Feline 15H05 3.3respectively. Similar to observations with antibody 11E12, the firstattempt at felinization of antibody 15H05 resulted in a loss of affinityto the feline IL-31 protein when compared to mouse 15H05 and a neutralaffect when compared to the 15H05 mouse feline chimera (FIG. 2, sectionsA and C). Similar to observations with felinized antibody 11E12 bindingto canine IL-31, certain combinations of feline 15H05 VH and VLframeworks had a neutral to positive impact on affinity to canine IL-31(See FIG. 2, section C Feline 15H05 1.1, 2.2, and 3.2).

In an effort to restore the affinity of felinized antibody 15H05, eachfelinized 15H05 VH was paired with the mouse 15H05 VL to generateheterochimeric antibodies. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1)the corresponding nucleotide sequence for which is (SEQ ID NO: 122;FEL_15H05_VH1) was combined with MU_15H05_VL (SEQ ID NO: 69;MU_15H05_VL) the corresponding nucleotide sequence for which is (SEQ IDNO: 70; MU_15H05_VL) to generate Feline 15H05 VH1 mouse VL.FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2) the correspondingnucleotide sequence for which is (SEQ ID NO: 124; FEL_15H05_VH2) wascombined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL) the correspondingnucleotide sequence for which is (SEQ ID NO: 70; MU_15H05_VL) togenerate Feline 15H05 VH2 mouse VL. FEL_15H05_VH3 (SEQ ID NO: 125;FEL_15H05_VH3) the corresponding nucleotide sequence for which is (SEQID NO: 126; FEL_15H05_VH3) was combined with MU_15H05_VL (SEQ ID NO: 69;MU_15H05_VL) the corresponding nucleotide sequence for which is (SEQ IDNO: 70; MU_15H05_VL) to generate Feline 15H05 VH3 mouse VL. Thesefelinized VH mouse VL heterochimeras were analyzed for their affinity tocanine and feline IL-31. Pairing of felinized 15H05 VH1 and VH3 withmouse 15H05 VL restored the affinity to feline IL-31 to equivalent orbetter than the mouse and chimeric forms. This trend in improvedaffinity was also observed to the canine IL-31 protein (FIG. 2, sectionsA and C).

To further dissect the positions in the 15H05 frameworks responsible foraffinity loss, a single felinized VH of 15H05 (FEL_15H05_VH1) was usedto pair with individual framework substitutions from mouse 15H05 VL.FEL_15H05_VH1 (SEQ ID NO: 122; FEL_15H05_VH1) the correspondingnucleotide sequence for which is (SEQ ID NO: 123; FEL_15H05_VH1) wascombined independently with FEL_15H05_VL1_FW1 (SEQ ID NO: 133;FEL_15H05_VL1_FW1) the corresponding nucleotide sequence for which is(SEQ ID NO: 134; FEL_15H05_VL1_FW1), FEL_15H05_VL1_FW2 (SEQ ID NO: 135;FEL_15H05_VL1_FW2) the corresponding nucleotide sequence for which is(SEQ ID NO: 136; FEL_15H05_VL1_FW2), and FEL_15H05_VL1_FW3 (SEQ ID NO:137; FEL_15H05_VL1_FW3) the corresponding nucleotide sequence for whichis (SEQ ID NO: 138; FEL_15H05_VL1_FW3) to create Feline 15H05 1.1 FW1,Feline 15H05 1.1 FW2, and Feline 15H05 1.1 FW3 respectively.Substitution of mouse 15H05 FW1 onto Feline 15H05 1.1 was detrimental tothe affinity to both feline and canine IL-31, however, when mouse FW2 orFW3 were substituted on Feline 15H05 1.1, excellent affinity wasachieved to canine and feline IL-31 with the FW2 being superior for bothspecies (FIG. 2, section C). Additional pairwise framework substitutionswere performed to determine the extent of affinity modulation by thisapproach. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1) thecorresponding nucleotide sequence for which is (SEQ ID NO: 122;FEL_15H05_VH1) was combined independently with FEL_15H05_VL1_FW1_2 (SEQID NO: 139; FEL_15H05_VL1_FW1_FW2) the corresponding nucleotide sequencefor which is (SEQ ID NO: 140; FEL_15H05_VL1_FW1_FW2),FEL_15H05_VL1_FW2_3 (SEQ ID NO: 143; FEL_15H05_VL1_FW2_FW3) thecorresponding nucleotide sequence for which is (SEQ ID NO: 144;FEL_15H05_VL1_FW2_FW3), and FEL_15H05_VL1_FW1_3 (SEQ ID NO: 141;FEL_15H05_VL1_FW1_FW3) the corresponding nucleotide sequence for whichis (SEQ ID NO: 142; FEL_15H05_VL1_FW1_FW3) to give Feline 15H05 1.1FW1_2, Feline 15H05 1.1 FW2_3, and Feline 15H05 1.1 FW1_3 respectively.Interestingly, the substitution of mouse FW1 alone was detrimental toaffinity while combinations of FW1 with FW2 or FW3 resulted in goodaffinity to both feline and canine IL-31 (FIG. 2, section C).

Finally, an attempt was made to minimize the number of backmutations inthe feline frameworks beginning with the most promising combinations offelinized VH and VL sequences. For this, FEL_15H05_VH1 (SEQ ID NO: 121;FEL_15H05_VH1) the corresponding nucleotide sequence for which is (SEQID NO: 122; FEL_15H05_VH1) was combined independently withFEL_15H05_VL1_FW2_K42N (SEQ ID NO: 145; FEL_15H05_VL1_FW2_K42N) thecorresponding nucleotide sequence for which is (SEQ ID NO: 146;FEL_15H05_VL1_FW2_K42N), FEL_15H05_VL1_FW2_V43I (SEQ ID NO: 147;FEL_15H05_VL1_FW2_V43I) the corresponding nucleotide sequence for whichis (SEQ ID NO: 148; FEL_15H05_VL1_FW2_V43I), FEL_15H05_VL1_FW2_L46V (SEQID NO: 149; FEL_15H05_VL1_FW2_L46V) the corresponding nucleotidesequence for which is (SEQ ID NO: 150; FEL_15H05_VL1_FW2_L46V),FEL_15H05_VL1_FW2_Y49N (SEQ ID NO: 151; FEL_15H05_VL1_FW2_Y49N) thecorresponding nucleotide sequence for which is (SEQ ID NO: 152;FEL_15H05_VL1_FW2_Y49N), and FEL_15H05_VL1_FW2_K42N V43I (SEQ ID NO:153; FEL_15H05_VL1_FW2_K42N_V43I) the corresponding nucleotide sequencefor which is (SEQ ID NO: 154; FEL_15H05_VL1_FW2_K42N_V43I) to giveFeline 15H05 1.1 K42N, Feline 15H05 1.1 V43I, Feline 15H05 1.1 L46V,Feline 15H05 1.1 Y49N, and Feline 15H05 1.1 K42N_V43I respectively.While the substitution of the entire mouse FW2 framework onto Felinized15H05 VL1 resulted in an antibody with excellent affinity to canine andfeline IL-31 (FIG. 2, section C, Feline 15H05 1.1 FW2), the individualbackmutations of FW2 amino acid residues had a neutral or detrimentaleffect indicating all 4 substitutions are necessary to maintain theoptimal tertiary structure for positioning of the CDRs on the IL-31epitope. Increased affinity of felinized 15H05 1.1 FW2 to feline andcanine IL-31 lead to the selection of this antibody for further work.

FIG. 5A shows an alignment of mouse antibody 11E12 VL sequence comparingpreviously referenced caninized 11E12 sequence to the felinizedversions. Noted below the alignment are dots showing the positons ofrelevant changes to Fel_11E12_VL1 that were necessary to restoreaffinity of this antibody to the IL-31 protein. Likewise FIG. 5B showsthe necessary changes to the felinized 15H05 VL (Fel_15H05_VL1) thatwere required to not only restore, but improve, its affinity to canineand feline IL-31 when compared to the mouse and chimeric forms of thisantibody.

1.9. Generation of Cell Lines Expressing Felinized Anti IL-31 Antibodiesfrom Glutamine Synthetase (GS) Plasmids

Felinized 15H05 1.1 FW2 was chosen as a candidate for the generation ofstable cell lines that will produce a homogenous supply of the antibodyfor further characterization. The genes encoding the felinized heavy andlight chains for cell line production were cloned into GS plasmids pEE6.4 and pEE 12.4 respectively (Lonza, Basel, Switzerland). The resultingplasmids were digested according to the manufacturer's protocol andligated together to form a single mammalian expression plasmid. ForZTS-927, the heavy chain is (SEQ ID NO: 121; FEL_15H05_VH1) thecorresponding nucleotide sequence for which is (SEQ ID NO: 122;FEL_15H05_VH1) combined with feline IgG heavy chain constant (SEQ ID NO:171; Feline_HC_AlleleA_wt) the corresponding nucleotide sequence forwhich is (SEQ ID NO: 172; Feline_HC_AlleleA_wt). For ZTS-927, the lightchain is (SEQ ID NO: 135; FEL-15H05-VL1_FW2) the correspondingnucleotide sequence for which is (SEQ ID NO: 136; FEL-15H05-VL1_FW2)combined with feline IgG light chain constant (SEQ ID NO: 175;Feline_LC_Kappa_G_minus) the corresponding nucleotide sequence for whichis (SEQ ID NO: 176; Feline_LC_Kappa_G_minus). For ZTS-361, the heavychain is (SEQ ID NO: 121; FEL_15H05_VH1) the corresponding nucleotidesequence for which is (SEQ ID NO: 122; FEL_15H05_VH1) combined withfeline IgG heavy chain constant (SEQ ID NO: 173; Feline_HC_AlleleA_1)the corresponding nucleotide sequence for which is (SEQ ID NO: 174;Feline_HC_AlleleA_1). For ZTS-361, the light chain is (SEQ ID NO: 135;FEL-15H05-VL1_FW2) the corresponding nucleotide sequence for which is(SEQ ID NO: 136; FEL-15H05-VL1_FW2) combined with feline IgG light chainconstant (SEQ ID NO: 175; Feline_LC_Kappa_G_minus) the correspondingnucleotide sequence for which is (SEQ ID NO: 176;Feline_LC_Kappa_G_minus).

To demonstrate transient production of antibody, each plasmid was usedto transfect HEK 293 cells and expression was carried out in varioussize cultures. Protein was isolated from conditioned HEK medium usingProtein A affinity chromatography according to standard proteinpurification methods. Medium was loaded onto chromatographic resin andeluted by pH shift. Eluted protein was pH adjusted, dialyzed, andsterile filtered prior to use. ZTS-361 was subsequently used forevaluation in the cat pruritus model to evaluate in vivo efficacy.Antibodies produced from a single GS plasmid, ZTS-927 and ZTS-361, weretested for affinity and potency. FIG. 2, section D shows the results forthe affinity assessment of these antibodies using biacore. The affinityof ZTS-927 and ZTS-361 to feline IL-31 is highly consistent with that ofthe mouse and chimeric form of the progenitor mouse mAb 15H05. Thepotency of these two antibodies was determined against canine and felineIL-31 using both canine and feline cell assays (FIG. 3). Consistent withprevious observations the IC50 values were proportionally higher whenusing the canine form of IL-31 with both cell types. The IC50 values forZTS-927 and ZTS-361 against feline IL-31 were also highly consistentwith values derived from the chimeric and mouse form of the antibodyindicating the final felinized version of mAb 15H05 produced form asingle GS plasmid was suitable for cell line development.

For generation of a stable cell line producing candidate antibodies, theGS plasmid was linearized prior to transfection with the restrictionenzyme, Pvul, which cuts at a single site in the plasmid backbone.GS-CHOK1SV (clone 144E12) cells were transfected with linearized plasmidDNA via electroporation. Following transfection, cells were plated in48-well plates (48WP) in order to generate stable pools. When pools wereat least 50% confluent in the 48WPs, 100 μl of supernatant was analyzedfor IgG expression using the ForteBio Octet and protein A biosensors(Pall ForteBio, Fremont, Calif.). The best expressing clones were scaledup into 6 well-plates (6 WP) and then into 125 mL shake flasks (SF).Once cells adapted to suspension culture in 125 mL flasks, 2 vials ofeach cell line pool were banked for LN storage. Since manufacturing celllines must be clonal, the top 3 highest expressing pools were subclonedby limiting dilution in 96 well culture plates. In order to proveclonality and avoid a second round of limiting dilution, 96 well plateswere imaged using Molecular Devices Clone-Select Imager (CSI) (MolecularDevices LLC, San Jose, Calif.) which captures images of single-cells andtheir subsequent growth. Clones were selected based on successful CSIimages, growth and production in 96WPs.

In order to assess cell culture growth and productivity, the topexpressing pools were further evaluated in a 14-day fed batch in 125 mLSFs. Cells were seeded in platform media and feeds consisting of LifeTechnologies' CD CHO plus 4 amino acids, proprietary feed CDF v6.2, and10% glucose. Following the 14-day Fed-Batch, pools were centrifuged andthe CD CHO produced mAB was isolated by filtering the supernatant via a0.20 μm Polyethersulfone (PES) membrane prior to purification.

A typical purification consists of two liters of conditioned medium(from CHO cell culture, 0.2 μm filtered) loaded onto a 235 mL column ofMabSelect (GE healthcare, cat #17-5199-02). The column had beenpre-equilibrated with PBS. The sample was loaded at a residence timeof >2.5 minutes. Following load, the column was washed again with PBS,and then with 25 mM sodium acetate, pH neutral. The column was elutedwith 25 mM acetic acid, pH 3.6, and then stripped with 250 mM aceticacid, 250 mM sodium chloride, pH˜2.2. Fractions (50 mL) were collectedduring the elution and strip steps. UV absorbance at A280 was monitoredthroughout. Peak fractions were pooled, pH adjusted to ˜5.5 with theaddition of 20 mM sodium acetate, and then dialyzed against threeexchanges of buffer. The dialysate was collected, sterile filtered, andstored at 4° C.

1.10. Identification of the Epitope on IL-31 Recognized by Antibody15H05

Knowledge of the epitope on IL-31 that is recognized by an antibody iscritical to understanding the mechanism by which it neutralizes thecytokine from binding to the IL-31Ra: OSMR co-receptor. In addition,knowing the epitope enables (but is not limited to) optimization ofantibody binding affinity and design of peptide epitope mimetics(mimotopes) which can have great utility as analytical capture reagentsand as a subunit vaccines to elicit a relevant focused immune response.A multistep process using CLIPS (Chemical Linkage of Peptides ontoScaffolds) technology (Timmerman et al. J Mol Recognit. 2007; 20(5):283-299) was used to identify and optimize a peptide capable of bindingto the paratope of mAb 15H05 (Pepscan, Lelystad Netherlands). Theaffinity of mAb 15H05 to both canine and feline IL-31 proteins is high(FIG. 2, MU-15H05) so the primary sequence of both IL-31 species wasconsidered relevant to this effort. A peptide microarray libraryrepresenting the canine IL-31 protein was created and used to identifypeptides capable of binding mAb 15H05 using an indirect ELISA. Followingidentification of peptides whose primary amino acid sequences representthe binding region of mAb 15H05 on IL-31, a focused full replacementanalysis was performed using peptides representing a segment of IL-31and replacing each of the 12 amino acids in this mAb 15H05 bindingregion with the 19 other possible amino acid residues at each position.This analysis was essential to identify the key amino acid residues onIL-31 involved with mAb 15H05 binding and also demonstrated wheresubstitutions on the canine primary sequence lead to an enhancement ofantibody binding.

The amino acids on the canine IL-31 protein that are recognized byantibody 11E12 were described previously (U.S. Pat. No. 8,790,651 toBammert, et al.). Therein were described the mutational analysis of thecanine IL-31 protein showing positions on the canine IL-31 protein thataffect binding of mAb 11E12 when converted to alanine. Based on the fullreplacement analysis described for mAb 15H05 above and previousknowledge of the binding epitope of 11E12, mutant forms of the felineIL-31 protein were created by substituting alanine for two key residueson the epitope recognized by each antibody (mutants described in section1.2 above). Mutations for each epitope were named according to theantibody that recognizes the site of the mutation (mutant 11E12 and15H05 vs. the native wt protein sequence).

FIG. 6A shows the alignment of wildtype feline IL-31 (SEQ ID NO: 157)with mutants 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161)highlighting the positions where the alanine substitutions occur. IL-31belongs to the IL-6 family of cytokines with the four helical bundlespossessing an up and down architecture (CATH database, Dawson et al.2017 Nucleic Acids Res. 2017 Jan. 4; 45 (Database issue): D289-D295). Ahomology model was generated based on the human IL-6 structure 1P9M(Boulanger et al. 2003 Science. June 27; 300(5628):2101-4) using the MOEsoftware (Chemical Computing Group, Montreal, QC, Canada). FIG. 6B showsthe feline IL-31 homology model highlighting the positions of the aminoacids involved with binding of antibodies 11E12 (site 1) and 15H05 (site2). The binding sites for each antibody appear to be located at separatepositions on the IL-31 protein.

To determine the impact of mAbs 11E12 and 15H05 ability to bind thesemutant forms of feline IL-31, an indirect ELISA was run using themutants directly coated on to an immunoassay plate. FIG. 6C shows theresults for this ELISA demonstrating that mAbs 11E12 and 15H05 arecapable of binding to wiltype feline IL-31 in this assay format. Whenmutant 11E12 is used as a capture protein, binding of mAb 11E12 ishighly attenuated and binding of mAb 15H05 is partially attenuated.Previous analysis of the 11 E12 epitope on canine IL-31 (described inU.S. Pat. No. 8,790,651 to Bammert et al.) indicated that 4 amino acidresidues impact binding of the mAb when mutated to alanine so themutation of 2 residues, in this case, may not be enough to completelyeliminate the high affinity binding of mAb 11E12 using this ELISAformat. The minor attenuation of mAb 15H05s binding to the 11E12 mutantis likely due to translational effects of the mutations from movement ofthe two front helices effecting the 15H05 binding site. The mutationsdesigned to affect mAb 15H05s binding (mutant 15H05) show a completeloss of mAb 1505s ability to bind this IL-31 mutant by ELISA. Unlike the11E12 mutant, the changes in the random coil recognized by mAb 15H05(mutant 15H05) had no impact on mAb 11E12 binding further supporting thedistinction between the two epitopes (FIG. 6C).

1.11. Competition Binding Assessment of mAbs 15H05 and 11E12 UsingBiacore

To further characterize the IL-31 epitopes bound by mAbs 15H05 and 11E12, blocking experiments were performed using biacore where the surfacecontaining the IL-31 protein was generated followed by sequentialaddition of antibodies. FIGS. 7A and 7B shows the relative binding ofeach antibody to canine IL-31 or feline IL-31, respectively, followingcapture of 11E12 or 15H05. The columns labeled HBS-EP (assay buffer)indicate the maximum signal obtained from each antibody binding to theIL-31 surface alone without competition. FIG. 7A shows the competitionbinding data for mouse 15H05 and 11E12 antibodies to canine IL-31. Theseresults clearly indicate that antibodies 15H05 and 11E12 are capable ofbinding to canine IL-31 in the presence of one another indicating theyrecognized distinct epitopes on the protein. The sensograms related toFIG. 7A show the disassociation kinetics of both antibodies are veryslow on this newly formed biacore surface therefore no additionaloccupation of binding sites can occur with addition of the same antibody(data not shown).

FIG. 7B shows the competition binding data for antibodies 15H05 and11E12 on a feline IL-31 surface again showing no overlap in the epitoperecognized. Binding of additional antibody in the presence of the sameantibody is a result of the increased off rate due to the poorer qualityof the surface used. Increased off rates can be seen and compared to theKD values from newly formed feline IL-31 surfaces in FIG. 2.

These results further support the epitope mapping data in section 1.10indicating a distinct epitope is recognized by the CDRs contained inantibody MU-15H05 when compared to MU-11E12. The epitope recognized byantibody 15H05 is distinct from antibody 11E12 described in (U.S. Pat.No. 8,790,651 to Bammert, et al.) and is a novel target on the IL-31protein for neutralization of this cytokine's activity in multiplespecies. These findings highlight the distinct spatial relationship ofbinding sites described in the feline IL-31 homology model (FIG. 6B) andsupport the hypothesis that this face of the cytokine is critical forinteraction with the IL-31Ra:OSMR receptor complex.

1.12. Synthesis and Characterization of Soluble Feline IL-31 Co-Receptor(IL-31RA and OSMR)

The human IL-31 heteromeric receptor co-complex, consisting of IL31Raand OSMR subunits, was shown to be required for IL31-mediatedintercellular activation of the JAK-STAT pathway and having involvementin atopic skin disease (Dillon et al. 2004 Nat Immunol. July;5(7):752-60, Dreuw et al. 2004 J Biol Chem. 279:36112-36120; and Diveuet al. 2004 Eur Cytokine Netw. 15:291-302). The human IL-31 Ra subunitwas later described as the initial binding event that occurs when IL-31is in contact with cell surface receptors and this event is apre-requisite for the recruitment of OSMR with subsequent formation of ahigh affinity co-receptor complex (Le Saux et al. 2010 J Biol Chem.January 29; 285(5):3470-7). We describe here evidence that the felineIL-31 protein is capable of binding to both OSMR and the IL-31 Raindependently. This observation is novel and has important implicationsto understanding how the IL-31 protein interacts with the IL-31Ra:OSMRco-receptor and to the biological role of IL-31 as it interactsindependently with individual subunits.

To enable understanding of how IL-31 binds to its co-receptor and tocharacterize the inhibitory properties of identified antibodies, tworeceptor forms were synthesized. The individual IL-31 receptor subunitIL-31Ra (SEQ ID NO: 169; Feline_IL31Ra_HIgG1_Fc_X1_Fn3) thecorresponding nucleotide sequence for which is (SEQ ID NO: 170;Feline_IL31Ra_HIgG1_Fc_X1_Fn3), and OSMR- (SEQ ID NO: 167;Feline_OSMR_hIgG1_Fc) the corresponding nucleotide sequence for which is(SEQ ID NO: 168; Feline_OSMR_hIgG1_Fc) were both constructed as humanIgG1 Fc fusions. By homology to the human homologs, the cytokinebinding, fibronectin III, and Ig-like domains were identified. Toevaluate the individual receptor subunits, the extracellular domains ofOSMR and the IL-31Ra (with its expected N-terminal proximal fibronectinIII domain) were generated as human IgG1 Fc fusions, both employingtheir native signal peptides. All synthetic cassettes were cloned intopcDNA3.1, expressed in the ExpiCHO system and purified as describedabove.

To analyze the ability of these receptor forms to bind wildtype andmutant IL-31 proteins, and indirect ELISA was run by coating 100 ul ofeach respective protein on an immulon 2HB plate (1 μg/ml) overnight incarb/bicarb buffer (sigma C3041-100CAP) at 4C. The ELISA plates werethen blocked with 5% NFDM blocking buffer in PBST for 1 hour at roomtemperature followed by binding of multiple concentrations of eachreceptor construct at room for 1 hour. Following washing with PBST, thepresence of the bound receptor (Fc fusion) was identified using mouseanti-human IgG1 (Lifetech A10684, 1:500 dilution) for 1 hour at roomtemperature. The wells were again washed with PBST and developed withKPL sureblue 3,3′,5,5′-tetra-methylbenzidine (TMB) microwell substrate.FIG. 8 shows the results for this indirect ELISA using wildtype andmutant forms of the feline IL-31 protein as a capture. These datademonstrate the ability of the wildtype feline IL-31 to independentlybind to the IL-31Ra and OSMR receptor subunits. These observations arein contrast to previous reports indicating the IL-31 protein initiallybinds to the IL-31Ra subunit and further recruits OSMR to the site. Asthe biological role of IL-31 is still being determined, it is of greatimportance to understand the dynamics of receptor binding and thepotential consequences to attenuation of its role in diseases such asatopic dermatitis. For this reason, consideration was further given tothese observations when characterizing antibodies the bind to epitopescapable of disrupting the ability of IL-31 to recognize IL-31Ra andOSMR.

In section 1.2 we describe the attenuated binding of antibodies 11E12and 15H05 to mutants with key amino acids in their binding sitesconverted to alanine (mutant 11E12 and 15H05 respectively). It wastherefore of great interest to understand the impact of these mutationson the ability to bind to the individual IL-31Ra and OSMR receptorsubunits. FIG. 8 shows that mutation in either the 11E12 or 15H05binding site completely disrupts IL-31Ra and OSMRs ability to bindindicating both antibodies bind epitopes that are necessary forinteraction of IL-31 with both receptor subunits. Lack of binding couldalso be due to changes in the confirmation of IL-31 resulting frommutation however these mutants are still capable of binding to antibodywhich suggests this is not the case. This key finding supports theability of both antibodies 11E12 and 15H05 (and derivatives) recognizingepitopes on IL-31 that neutralize the cytokine's ability to signalthrough its co-receptor and further block cell association of thecytokine to either receptor during this process. These data support theidentification of antibodies that are capable of removing IL-31 fromcirculation and rendering it unable to bind to cell surface or solublereceptor forms.

1.13. In Vivo Evaluation of Chimeric Antibodies in a Feline IL-31Pruritus Challenge Model

The ability of an antibody to effectively neutralize its target can beassessed in vitro through examination of binding to a relevant epitopeon the target protein with the appropriate affinity and potency in acell based assays that allow extrapolation to in vivo potency. Describedabove are the steps taken to characterize two series of antibodiesgenerated from the mouse progenitor mAbs 11E12 and 15H05. Section 1.7describes the generation of mouse: feline chimeric forms of mAbs 11E12and 15H05 with a resulting affinity to canine and feline IL-31 that arecomparable to the original mouse monoclonal antibody (FIG. 2, sectionA). The mouse: feline chimeric forms of 11E12 and 15H05 also hadcomparable IC50 values showing inhibition of feline IL-31 induced pSTAT3signaling in canine and feline macrophage cells (FIG. 3). During thefelinization process in section 1.8, mouse mAb 11E12 was converted tothe felinized version (Feline 11E12 1.1) with subsequent loss ofaffinity to canine and feline IL-31 (FIG. 3) and loss of potency againstfeline IL-31 signaling in canine and feline cells (FIG. 3). Prior tooptimization of the felinized 11E12 and 15H05 antibodies described insection 1.8, it was of interest to understand the ability of thesepreliminary felinized and chimeric forms to neutralize the pruriticactivity of feline IL-31 in a cat challenge model. Of interest was thepharmacodynamic effect of these different antibodies on neutralizationof pruritus and to understand any correlation to affinity, cellularpotency, or epitope recognition that may influence efficacy. Goingforward a range of cellular potency that correlates to in vivo efficacyin the pruritus challenge model could be predictive of furtheroptimization necessary using in vitro assays.

To test the preliminary efficacy of mouse: feline 11E12 chimera, mouse:feline 15H05 chimera, and felinized 11E12 (Feline 11E12 1.1), an IL-31induced pruritus model in cats was developed. Following an intravenousdose of 0.5 μg/kg feline IL-31 (SEQ ID NO: 159; Feline_IL-31_E_coli),the corresponding nucleotide sequence for which is (SEQ ID NO: 160;Feline_IL-31_E_coli), cats will portray transient pruritic behavior thatincludes (but is not limited to) licking, chewing, scratching, and heador body shaking. Rubbing up against the cage was not considered apruritic activity. Pruritic observations take place by a trainedinvestigator for 30 minutes prior to administration of the IL-31 proteinand for 1 hour following. For this study, a baseline challenge withfeline IL-31 was performed up to 1 month prior to dosing with antibody.On day zero, a 0.5 mg/kg antibody dose was combined with 0.5 μg/kg offeline IL-31 at room temperature for 60 minutes prior to injecting thepre-bound complex into each animal. A “no mAb” control was included fora control. The dose of mAb represents a gross molar excess of antibodyto cytokine. Pruritic activity was monitored as described on days 0, 7,and 21. Results in FIG. 9 show significant improvement (p<0.05) inpruritus scores with mAb mouse: feline 15H05 chimera at days 0, 7, and21 when compared to the placebo control. Although the mouse: feline11E12 chimera showed an initial trend in efficacy at day zero, it didnot achieve a significant reduction in pruritus at any timepoint whencompared to vehicle placebo. Feline 11E12 1.1 did not reduce pruritus atday zero and showed no trend in efficacy when compared to vehicleplacebo so further IL-31 challenges on days 7 and 21 were not performed.

Taken together these results show a clear delineation between theactivities of these antibodies with the lack of efficacy for feline11E12 1.1 at preventing pruritic behavior in the cat induced by IL-31.The loss of affinity and potency of feline 11E12 1.1 likely resulted inthe lack of in vivo efficacy. When comparing the efficacy outcome ofmouse: feline 11E12 chimera and mouse: feline 15H05 chimera thedistinction is more subtle. The chimeric forms of both mAbs have acomparable KD value to their mouse progenitor with the affinity ofmouse: feline 11E12 being slightly superior to both feline and canineIL-31 (FIG. 2, section A). This increased affinity however does nottranslate directly to increased potency as the mouse: feline 15H05chimera has an approximately 2-fold increased IC50 to that of mouse:feline 11E12 chimera against feline IL-31 induced pSTAT3 signaling infeline FCWF4 cells (FIG. 3). These data suggest that the manner in whichantibody 15H05 CDRs recognize feline IL-31 is superior at neutralizingthe cytokine's ability to signal through its co-receptor in turn makingit more effective at blocking pruritus in cats. The differences in IC50sobserved in these cellular assays offers a promising means to predict invivo potency and to discriminate subtle differences in epitoperecognition both within and between series of antibodies.

1.14. In Vivo Evaluation of the Efficacy of Felinized 15H05 Anti IL-31Antibodies in a Cat Pruritus Challenge Model

Based on the positive efficacy outcome using the mouse: feline 15H05chimera described above, further work was done to increase the affinityand potency of felinized 15H05 (described above in section 1.8).Systematic substitution of the variable light chain feline frameworks inantibody feline 15H05 1.1 lead to the identification of Feline 15H05 1.1FW2 having increased affinity to both feline and canine IL-31 comparedto mouse 15H05 (FIG. 2). Combination of the heavy and light chains ofFeline 15H05 1.1 FW2 into a single plasmid led to the formation ofZTS-927 and ZTS-361 antibodies following production from HEK and CHOexpression systems. The affinity and potency of both antibodiesresulting from expression from a single plasmid are also described inFIGS. 2 and 3, respectively.

The efficacy of the fully felinized anti feline IL-31 mAb ZTS-361 wasassessed for its ability to neutralize pruritic behavior in an IL-31induced in vivo cat model. FIG. 10A shows the baseline pre-challengepruritic behavior for the T01 vehicle placebo and T02 antibody ZTS-361groups from day −7 through day 28 with day zero being the day ofantibody administration to group T02. As shown in this graph, thevariance of pruritic behavior scored for both T01 and T02 groups priorto IL-31 challenge varied little with the number of pruritic eventsobserved between 0 and 10 within the 30 minute observation period. Thisstudy differed from the preliminary feline challenge model describedabove in section 1.13 in that on day zero cats were dosed with 4 mg/kgZTS-361 subcutaneously without combination with feline IL-31 to generatea pre-bound complex. This represents a more rigorous assessment ofefficacy as antibody ZTS-361 will be in circulation for seven days priorto the first IL-31 challenge requiring the antibody to have sufficientexposure to bind and neutralize circulating IL-31.

For this study, pruritic behavior was assessed on days 7, 21, and 28 for1 hour following a 0.5 μg/kg intravenous challenge of the IL-31 protein.FIG. 10B shows the efficacy of antibody ZTS-361 demonstrating asignificant reduction in pruritus observed on days 7 (p<0.0001), 21(p<0.0027), and 28 (p<0.0238) following IL-31 challenge when compared tovehicle placebo control. Data from this challenge model support previousobservations demonstrating the efficacy of mouse: feline 15H05 chimeraand support the cell-based potency and relevance of the epitope onfeline IL-31 recognized by the 15H05 CDRs. These data further supportthe ability of antibody ZTS-361 to neutralize pruritus induced by felineIL-31 in vivo and suggest this antibody may serve as a therapeutic inthe treatment of IL-31 mediated disease in cats including atopicdermatitis.

Recent data examining the plasma levels of IL-31 in client owned animalsshows an increased amount of the cytokine in circulation among dogs withatopic and allergic dermatitis compared to normal laboratory beagles(FIG. 11A). A recent study was performed to determine serum IL-31 levelsin cats with a presumptive diagnosis of allergic dermatitis (AD) fromseveral different geographic regions in the USA. FIG. 11B shows theresults from this assessment indicating that, like dogs with atopic andallergic dermatitis, 73 cats surveyed with this presumptive diagnosishad mean circulating IL-31 levels of 8799 fg/ml compared to 205 fg/ml inthe 17 age-matched control cats. To understand the levels of canineIL-31 in a previous model development study, the pharmacokinetic profileof canine IL-31 was analyzed in dogs following administration of asubcutaneous dose of 1.75 μg/kg. FIG. 110 shows peak plasma levelswithin the first hour reaching a maximum of about 30 ng/ml and amaintained level of about 400 μg/ml at three hours. Based on thesefindings it is reasonable to believe that an intravenous dose of 0.5μg/kg feline IL-31 used in this feline model will result in acirculating amount that is far excessive to that observed in thenaturally occurring disease state for dogs and cats.

2. Example 2 Characterization and Use of IL-31 Mimotopes in Vaccines andin Diagnostics

2.1. Amino Acid Residues on Canine IL-31 that are Involved with Antibody15H05 Binding

As described in section 1.10 of this application, a full replacementscan of the canine IL-31 protein was performed encompassing the aminoacids outlined in FIG. 12. Each position described within this sectionof IL-31 was individually replaced in the full-length protein with oneof the other possible 19 amino acids and binding of antibody 15H05 wasassessed using an indirect ELISA. Those substitutions having no impacton binding resulted in an ELISA signal equivalent to (or higher) thanthe with IL-31 protein, while substitutions impacting antibody bindingresulted in a lower (or no) signal from the assay. As detailed in FIG.12, some positions on canine IL-31 were tolerant of certainsubstitutions of the amino acids indicated (SEQ ID NO: 155; positions124, 125, 129, and 132-135) while others were not (SEQ ID NO: 155;positions 126, 127, 128, 130, and 131). For comparison, thecorresponding region on feline (SEQ ID NO: 157), equine (SEQ ID NO:165), and human IL-31 (SEQ ID NO: 181) are shown. One can extrapolatethe mutational observations in canine IL-31 to the other species fordesign of homologous peptides. This fine positional mapping of theepitope region on canine IL-31 allowed for the design of both linear andconstrained peptides that mimic the binding site on the IL-31 proteinrecognized by antibody 15H05. These results in conjunction with thefeline IL-31 model described in section 1.10 (FIG. 6B) indicate theepitope recognized by mAb 15H05 is a consecutive region of amino acidsthat form the convergence of a random coil leading into the fourthhelical domain of the cytokine. Presentation of this epitope allowsbinding of mAb 15H05 to both linear and constrained peptiderepresentations with greater affinity associated with the constrainedform. Mutational data described above (section 1.12) further emphasizesthe important positioning of this epitope (and the 11E12 epitopepreviously described in U.S. Pat. No. 8,790,651 to Bammert et al.) onthe face of the IL-31 protein with respect to binding to the co-receptorcomplex.

2.2. Characterization of Peptide Mimetics Representing the Epitope onIL-31 Recognized by Antibody 15H05

As mentioned previously, the design of peptide epitope mimetics(mimotopes) can have great utility as analytical capture reagents and assubunit vaccines to elicit a relevant focused immune response togenerate antibodies in an animal directed at a neutralizing epitope onIL-31. Towards this goal, canine and feline peptides were designed andcharacterized for their affinity to antibody ZTS-927 and their abilityto block antibody ZTS-361s ability to inhibit IL-31 mediated receptorsignaling on feline FCFW4 cells. The construction of antibodies ZTS-927and ZTS-361 (both containing CDRs from mouse antibody 15H05) aredescribed above in section 1.9. Peptide ZTS-561 contains the amino acidsequence N-TEISVPADTFERKSFILT-C which corresponds to positions 121through 138 of SEQ ID NO: 155 with the substitution of Arginine (R) forCysteine (C) at position number 132. Peptide ZTS-561 also includes N andC terminal Cysteines to facilitate conjugation chemistry using the freethiol groups. Peptide ZTS-562 contains the amino acid sequenceN-EISVPADTFERKSF-C which corresponds to positions 122 through 135 of SEQID NO: 155 with the substitution of Arginine (R) for Cysteine (C) atposition number 132. Peptide ZTS-562 also includes N and C terminalCysteines to facilitate conjugation chemistry using the free thiolgroups. Peptide ZTS-563 contains the amino acid sequenceN-AKVSMPADNFERKNFILT-C which corresponds to positions 121 through 138 ofSEQ ID NO: 157 with the substitution of Threonine (T) for Alanine (A) atposition number 138. Peptide ZTS-563 also includes N and C terminalCysteines to facilitate conjugation chemistry using the free thiolgroups. Peptide ZTS-564 contains the amino acid sequenceN-TEISVPADTFERKSFILT-C which corresponds to positions 121 through 138 ofSEQ ID NO: 155. Peptide ZTS-564 also includes N and C terminal Cysteinesto facilitate conjugation chemistry using the free thiol groups. Amultistep process using CLIPS technology (Timmerman et al. J MolRecognit. 2007; 20(5): 283-299) was used to identify and optimize thesefour peptides capable of binding to the paratope of mAb 15H05 (Pepscan,Lelystad Netherlands). For the purpose of generating immunogens, thesefour peptides (depicted in FIG. 13A) were independently conjugated to acarrier protein which is an inactive mutant (non-toxic) form ofdiphtheria toxin (CRM197) using standard cross-linking chemistry. Foraffinity assessment, each peptide was independently immobilized to abiacore surface and the KD for the felinized anti IL-31 15H05 mAb(ZTS-927) was determined (FIG. 13B). All four peptides bound ZTS-927with nanomolar affinity indicating they are close representations of thebinding site on full length IL-31. To assess the potency of eachpeptide, a dose titration of conjugated or unconjugated peptides wereco-incubated at 37° C. for 1 hour with 0.2 μM (6.5 μg/ml) of mAb ZTS-361prior to addition of feline IL-31 on FCWF-4 (feline macrophage-likecells). IC50 values were calculated using increasing concentrations ofpeptide (x-axis) versus the percent effect (y-axis) defined as theability of the peptide to bind and block mAb ZTS-361 inhibition of IL-31protein mediated STAT3 phosphorylation in feline FCWF-4 macrophages.Peptide ZTS-564 had reduced solubility in solution which likely resultedin inefficient conjugation, low epitope density, and poor potency.Peptide ZTS-561 had poor potency in the conjugated form but maintained agood potency when unconjugated (IC60˜1.7 μg/ml). ZTS-562 and ZTS-563both demonstrated excellent potency unconjugated with IC50s of 1.046μg/ml and 1.742 μg/ml respectively. The potency declined approximately3-fold following conjugation with IC50s for ZTS-562 and ZTS-563 of 3.024μg/ml and 3.384 μg/ml respectively (FIG. 13B). The ability of thesepeptides to block the high affinity binding of mAb ZTS-361 to the IL-31protein was highly promising and gave further evidence to support theirutility as epitope mimetics (further referred to as IL-31 15H05mimotopes) of a relevant epitope on IL-31. These IL-31 15H05 mimotopeswere further explored for their utility as immunogens to elicit ananti-IL-31 immune response.

2.3. Study Design for Generating Serum Titers to IL-31 FollowingImmunization of Beagle Dogs with IL-31 15H05 Canine and Feline Mimotopesand Full-Length Feline IL-31 Protein

An immunogenicity study was undertaken to assess the ability ofCRM-197-conjugated IL-31 15H05 mimotopes to generate an epitope-specificimmune response driven towards the relevant region on the IL-31 proteinwhere antibody 15H05 binds and neutralizes the cytokines ability toactivate the IL-31Ra:OSMR co-receptor. The study design is depicted inFIG. 14. Purebred male beagle dogs were subcutaneously administered theconjugated IL-31 15H05 mimotopes adjuvanted with ZA-01. The diagrambelow shows the experimental design by group. Control groups wereincluded containing ZA-01 adjuvant CRM-197 alone (T01) and CRM-197conjugated feline IL-31 (SEQ ID NO: 159; Feline_IL-31_E_coli), thecorresponding nucleotide sequence for which is (SEQ ID NO: 160;Feline_IL-31_E_coli) in ZA-01 (T02). 10 μg/dose of each adjuvantedmimotope or control was administered subcutaneously on days 0, 28, and56 (0.5 ml of a 20 μg/ml solution). Blood for serum was taken on day 0(pre-dose), 7, 12, 28 (pre-dose) 35, 42, 49, 56 (pre-dose), 63, 70, 77,and 84. In addition, on days 35 and 84, approximately 40 mls of bloodwas collected from each animal into lithium heparin tubes and processedfor PBMC isolation using a standard method. PBMCs were cryopreservedfollowing isolation until further evaluation of antigen-specificB-cells.

2.4. Serum Titers Generated Following Vaccination of Dogs with IL-3115H05 Canine and Feline Mimotopes and Full-Length Feline IL-31 Protein

Serum titers from each study day indicated in section 2.3 above wereassessed for each animal. Titers were determined using an indirect ELISAwhere a full-length IL-31 protein was used as the capture material foreach respective assay. Serum was assayed from each study group forbinding to feline, feline 15H05 mutant, canine, equine, and human IL-31proteins. The objective was to understand the immune response elicitedby feline IL-31 protein (SEQ ID NO: 159; Feline_IL-31_E_coli), or 15H05peptide mimotopes, against multiple species of IL-31 having a range ofamino acid sequence identities to one other (FIG. 1A). Treatment group 2(full-length Feline IL-31 CRM) represents the adaptive immune responseto multiple epitopes spanning the entire protein sequence. Using thefull protein as an immunogen will generate antibodies that are bothneutralizing and non-neutralizing to the bioactivity of IL-31. Previouswork in mice describing identification of neutralizing antibodies toIL-31 signaling indicates that the percentage of these antibodies aresmall and therefore the majority of the polyclonal response to thefull-length protein will be that of a non-neutralizing type (U.S. Pat.No. 8,790,651 to Bammert, et al.). As a vaccine approach, generation ofnon-neutralizing antibodies to IL-31 may have adverse effects on safetyand efficacy. Non-neutralizing antibodies may result in increasedamounts of bioactive IL-31 in circulation resulting from bound antibodycytokine complexes. These complexes can allow monomeric or aggregatedforms of IL-31 to exist in circulation allowing availability of thereceptor binding portion of the IL-31 to interact with the IL-31:OSMRco-receptor. Increased pSTAT signaling resulting from increased IL-31 incirculation will exacerbate pruritic activity in a disease state likeatopic dermatitis (Gonzales et al. Vet Dermatol. 2013 Febuary;24(1):48-53.e11-2).

FIGS. 15A-E shows the average titer results to each respective IL-31protein organized by treatment group showing the response at each dayserum was taken. Serum titers were examined to IL-31 using multiplespecies of the protein to understand the extent of Cross-ReactiveAntibody Response (CRAR) that may occur. The maximum dilution tested foreach serum sample was 1:50,000 so when the titer exceeded this value itwas designated as 50,000. For clarity, the following description offigures will proceed according to the individual treatment groupsresponse to each IL-31 protein used for the capture ELISA.

For dogs vaccinated with full-length feline IL-31 (T02), it was expectedthat the polyclonal serum generated would bind multiple species giventhe high percent identity between homologs. FIG. 15A shows the canineantibody titers generated that bind to feline IL-31. Analysis of the T02group shows a moderate and sustained antibody response was generated tothe full-length feline IL-31 protein following the third dose whichpersisted to the termination of the study at day 84. When examining theT02 group response against the feline 15H05 mutant protein (SEQ ID NO:163; Feline_IL31_15H05_mutant), a similar profile is seen with titerseven further elevated on day 63, 70, and 77 (FIG. 15B). FIG. 15C showsthe titers to canine IL-31. When looking at titers from the T02 group weexamine the extent to which the vaccinated dogs mounted a CRAR to thecanine IL-31 protein. No response was observed prior to the third doseof feline IL-31 CRM. Following dose 3 on day 56, a transient CRAR can beobserved from days 63-77 returning to near baseline by day 84. Themagnitude of the anti-canine response was similar to the anti-felineresponse however the duration of was shorter. Interestingly, the CRAR tohorse and human IL-31 was negligible to minor respectively (FIGS. 15Dand 15E, days 63 and 84 for human). In summary, the dog's immuneresponse to feline IL-31 CRM was most robust and persistent against thefeline IL-31 protein itself. Feline and canine IL-31 share a 76% aminoacid identity to each other which appears to be a sufficiently highenough level for a CRAR to occur to the canine protein. Horse and humanIL-31 have a 57 and 49% identity to feline respectively yielding only aminor CRAR in the case of human protein titers.

IL-31 15H05 mimotope ZTS-561 represents the binding site on canine IL-31recognized by antibody 15H05. Antibody responses from dogs vaccinatedwith ZTS-561 CRM are described as T03 in FIGS. 15A-E. The objective herewas to assess the antibody response to this specific region on IL-31known to be involved with the cytokine's interaction with its receptor.Focusing the immune response to a specific epitope will ensureantibodies are directed to an area on the protein that will result inneutralizing its biological activity. ZTS-561 CRM is a constrained20-mer representing the portion of the canine IL-31 protein recognizedby antibodies with identical CDRs to murine antibody 15H05 (SEQ ID NO:67; MU_15H05_VH), the corresponding nucleotide sequence for which is(SEQ ID NO: 68; MU_15H05_VH) paired with VL (SEQ ID NO: 69;MU_15H05_VL), the corresponding nucleotide sequence for which is (SEQ IDNO: 70; MU_15H05_VL). Mimotope ZTS-561 failed to produce a stronganti-feline IL-31 response throughout the entire study (FIG. 15A)including to the feline IL-31 15H05 mutant (FIG. 15B). In contrast, thedog's immune response to ZTS-561 CRM against the canine IL-31 proteinwas very strong beginning at day 35 following the second injection andpersisting through the termination of the study at day 84 (FIG. 15C).The CRAR elicited by ZTS-561 to equine IL-31 was negligible and to humanonly a small response was observed on day 56 of the study (FIGS. 15D and15E). It is interesting to note that even though the feline and canineproteins share a high degree of identity in this region of the protein(FIG. 1), a species-specific immune response was directed toward the dogIL-31 protein following vaccination of dogs with the canine 15H05mimotope.

ZTS-562 CRM is a constrained 16-mer truncated version of ZTS-561 againrepresenting the portion of the canine IL-31 protein recognized byantibodies with identical CDRs to murine antibody 15H05 (SEQ ID NO: 67;MU_15H05_VH), the corresponding nucleotide sequence for which is (SEQ IDNO: 68; MU_15H05_VH) paired with VL (SEQ ID NO: 69; MU_15H05_VL), thecorresponding nucleotide sequence for which is (SEQ ID NO: 70;MU_15H05_VL). Data for the dog's response to ZTS-562 are found as theT04 group in FIGS. 15A-E. Interesting the CRAR elicited by this shorterversion was more pronounced resulting in modest anti-feline titers ondays 35 through 70 (FIG. 15A). Some response was also observed to themutant 15H05 IL-31 protein between days 35 and 63 (FIG. 15B). Theanti-canine IL-31 response elicited by this mimotope was outstandingbeginning on day 35 following the second dose and persisting through thetermination of the study on day 84. Consistent with other results withcanine peptide ZTS-561 CRM, ZTS-562 CRM had no CRAR with the equine andhuman proteins.

ZTS-563 CRM is a constrained 18-mer and is the only mimotoperepresenting the portion of the feline IL-31 protein recognized byantibodies with identical CDRs to murine antibody 15H05 (SEQ ID NO: 67;MU_15H05_VH), the corresponding nucleotide sequence for which is (SEQ IDNO: 68; MU_15H05_VH) paired with VL (SEQ ID NO: 69; MU_15H05_VL), thecorresponding nucleotide sequence for which is (SEQ ID NO: 70;MU_15H05_VL). Data for the dog's response to ZTS-563 are found as theT05 group in FIGS. 15A-E. Consistent with previous observations of aspecies-specific response to the canine mimotope, the cat mimotope(ZTS-563) elicited a feline-specific anti-IL31 response in the dog. FIG.15A shows the anti-feline IL-31 titer response to vaccination withZTS-563 reaching greater than 1:50,000 on day 35 and sustained at thislevel through day 77 falling modestly at day 84. Comparing the T05(ZTS-563) treatment group between FIGS. 15A and 15B one can clearly seethe difference in titer between feline IL-31 and the feline IL-31 15H05mutant. The time dependent decrease in titer to the mutant protein (whencompared to the wildtype protein) indicates that a significant portionof the immune response is directed to a very specific portion of theprotein represented by mimotope ZTS-563. Remarkably, the anti-canineIL-31 response was modest to low, further supporting thespecies-specific response generated by the dog's immune system, tosubtle differences in the amino acid sequences of the two species.Vaccination with ZTS-563 is the only mimotope that generated a CRAR indog to the equine IL-31 protein. These observations demonstrate subtlechanges in the mimotope sequence can lead to species specificity and mayalso impart a cross-species immunogenic response. Understanding theseproperties is beneficial to the design of an IL-31 directed vaccine forsingle or multi-species use using this technology.

Lastly is ZTS-564 CRM, a constrained 18-mer identical to ZTS-561 howeverutilizing an alternate linker, mT2b (FIG. 15A). ZTS-564 CRM representsthe portion of the canine IL-31 protein recognized by antibodies withidentical CDRs to murine antibody 15H05 (SEQ ID NO: 67; MU_15H05_VH),the corresponding nucleotide sequence for which is (SEQ ID NO: 68;MU_15H05_VH) paired with VL (SEQ ID NO: 69; MU_15H05_VL), thecorresponding nucleotide sequence for which is (SEQ ID NO: 70;MU_15H05_VL). Data for the dog's response to ZTS-564 are found as theT06 group in FIGS. 15A-E. Consistent with other observations there islittle to no dog anti-feline IL-31 response elicited by this mimotope(FIG. 15A and 15B). The anti-canine IL-31 response generated by ZTS-564was very robust. FIG. 15C shows of all the treatment groups in thisstudy, T06 (ZTS-564) is the only one generating an immune responseagainst canine IL-31 following a single dose. The anti-canine IL-31titers generated following the second and third dose resulted in amaximal assay response (greater than 1:50000) at days 35 through thetermination of the study at day 84. There was no CRAR to equine IL-31observed however this mimotope produced the only consistent response tohuman IL-31 observed among the treatment groups. It is noteworthy thatsuch subtle differences in the linker chemistry and perhaps the moredefined constraints of the mT2b linker provide a more precise directedanti IL-31 response potentially alleviating the need for more frequentdosing.

Data from this study indicates that peptide mimetics representing thebinding site of the neutralizing anti-IL-31 antibody 15H05 are capableof eliciting an immune response in an animal and this immune response isdirected against the epitope recognized by the antibody 15H05 CDRs. Itis conceived from these data that further characterization of thisanti-serum using recombinant IL-31 co-receptors can be used to definethe IL-31 neutralizing fraction generated during this polyclonalresponse. These results further suggest the utility of such an approachfor use as a vaccine against an IL-31 mediated disorder like atopicdermatitis.

2.5. Identification of IL-31 Neutralizing Antibodies from IndividualB-Cells Isolated from Plasma Cells of Beagle Dogs Immunized with IL-3115H05 Mimotopes

As described above, blood was harvested on days 35 and 84 of the studyfor the purpose of isolating PBMCs. PBMCs from a single T05 dogvaccinated with ZTS-563 CRM (the feline mimotope) were used for furtherevaluation of antibody positive B-cells due to the robust 15H05epitope-directed response (FIGS. 15A and 15B). Activated memory B-cellswere screened for those cells secreting antibodies using an anti-canineIgG Fc antibody coupled to a bead.

Secreted IgGs were simultaneously assessed for their ability to bindwildtype feline IL-31 and bind to the feline IL-31 15H05 mutant. Theseprimary screening results lead to the selection of 7 hits from this PBMCcell population. Of these 7 hits, 3 did not bind to the 15H05 mutantindicating these B-cells are making antibodies with the closestrecognition of the 15H05 epitope as a result of immunization with theIL-31 15H05 mimotope ZTS-563 (data not shown). Following sequencing ofthe variable heavy and light chains for these 7 hits, recombinant fullycanine versions were constructed, expressed in HEK cells, and purifiedas previously described herein. Re-screening of these seven recombinantcanine IgGs resulted in only a single hit (ZIL1) that retained bindingto the feline IL-31 protein (FIG. 4). Furthermore, binding of ZIL1 tothe IL-31 15H05 mutant is decreased, using ELISA and Biacore methods,indicating this antibody binds to a common epitope region as antibody15H05. The additional hits derived directly from canine B-cells insection 1.6 and in FIG. 4 (ZIL8-ZIL171) were from dogs immunized withfull length feline IL-31 and from other tissue sources previouslydescribed herein. Only the ZIL1 antibody was derived from PBMCsfollowing vaccination with a peptide mimotope.

An important aspect of these findings is the ability to identify mAb15H05 epitope-specific antibody secreting B-cells in the circulation ofa dog following immunization with a peptide mimotope from the IL-31protein. These results validate the use of a peptide mimicking theepitope region on IL-31 known to be relevant to the mode of inhibitoryaction of antibody 15H05. As antibodies with CDRs derived from mAb 15H05are capable of preventing IL-31 mediated pruritus in vivo, these resultsfurther support the concept of immunization with such a mimetic designedto generate an epitope-focused immune response as a vaccine approach forthe prevention of IL-31 mediated diseases such as atopic dermatitis.

2.6. Use of IL-31 15H05 Mimotope as a Capture Reagent to Measure theIdentity and Potency of Antibody 15H05 and its Derivatives

An IL-31 15H05 mimotope represents the binding site on the IL-31 proteinthat is recognized by the CDRs from the mouse progenator mAb 15H05. Asmentioned previously it is desirable to have such a reagent to use in anELISA (or other) assay format to monitor the antibody production processthroughout manufacturing. It is conceived herein that such mimotopesdescribed would have such utility where a production lot of ananti-IL-31 antibody is made and a peptide mimotope would be used for ananalytical assay to verify the identity and quantity of antibodyproduced.

2.7. Use of IL-31 15H05 Mimotope as a Diagnostic to Measure AntibodyLevels or to Determine IL-31 Levels in a Host Species

It is further conceived herein the use of a peptide mimotope asdescribed to be used as an analytical assay reagent to measure theamount of circulating antibody in a host following treatment of atherapeutic for an IL-31 mediated disorder such as atopic dermatitis.Body fluid from an animal is added directly to the mimotope which isbound to a solid surface and then appropriate secondary detectionreagents are added to quantify the level of antibody. Additionally, anassay design is conceived here whereby a mimotope is used to capture anantibody that is labeled for detection in an assay. This capturedantibody would have an affinity to the attached mimotope that is lowerthat the affinity of native circulating IL-31 in a host species. In thisembodiment, incubation of the fluid derived from the host species isincubated with the labeled antibody: mimotope complex that is tetheredto a solid surface. The presence of IL-31 in the test fluid derived fromthe host species will have a higher affinity to the antibody, thusliberating the labeled antibody from the solid surface where it can beremoved during wash steps. The level of IL-31 in the test fluid can thusbe correlated to the lack of signal that appears on the mimotope-boundsurface. It is conceived that such an assay would have utility tomeasure IL-31 in a research or clinical setting for use as a diagnostictest.

2.8. Serum Titers to IL-31 Following Immunization of Beagle Dogs withIL-31 Mimotopes and Full Length Canine IL-31 Protein

A second serology study was performed using purebred neutered malebeagles like the study design described herein in section 2.3 however inthis study different mimotopes were compared. Purebred male beagle dogswere subcutaneously administered the conjugated IL-31 mimotopesadjuvanted with ZA-01. A control group was included containing CRM-197conjugated canine IL-31 (SEQ ID NO: 155; Canine_IL-31), thecorresponding nucleotide sequence for which is (SEQ ID NO: 156;Canine_IL-31) in ZA-01 (T01). 10 μg/dose of each adjuvanted mimotope orcontrol was administered subcutaneously on days 0, 28, and 56 (0.5 ml ofa 20 μg/ml solution). Blood for serum was taken on day −1, 0 (pre-dose),14, 28 (pre-dose), 42, 56 (pre-dose), 70, and 84. In addition, on days−1, 35 and 63, approximately 40 mls of blood was collected from eachanimal into lithium heparin tubes and processed for PBMC isolation usinga standard method. PBMCs were cryopreserved following isolation untilfurther evaluation of antigen-specific B-cells.

Treatment group 1 (full-length Canine IL-31 CRM) represents the adaptiveimmune response to multiple epitopes spanning the entire proteinsequence like the rationale described in section 2.4 using full lengthfeline protein. FIG. 16A shows a table outlining the study treatmentgroups. In addition to the full-length protein described for T01, twomimotopes representing the 15H05 epitope on canine (T02, ZTS-420) andhuman (T03, ZTS-421) were used. ZTS-420 is like ZTS-561 describedpreviously in the feline serology study however ZTS-420 is constrainedby a disulphide bond formed by cysteines at the N and C terminus of thepeptide compared to the mT2a linker found in ZTS-561. ZTS-421 is thehomologous region on the human IL-31 protein (SEQ ID NO: 181;Human_IL-31) constrained by linking the N and C termini with the mT2blinker described in FIG. 13A. Reference to the key amino acid sequencesinvolved with antibody 15H05 binding can be found in FIG. 12. A fourthgroup was included to explore the immunogenic potential of a keyantibody binding region on canine IL-31 previously described using twoantibodies known to neutralize IL-31 mediated pSTAT signaling and IL-31mediated pruritus in dogs (U.S. Pat. No. 8,790,651 to Bammert, et al.).This region on feline IL-31 is highlighted in the homology model shownin FIG. 6B. The accepted model of IL-31 is as a four-helical domaincytokine with the helices forming an alternating up and down topology.For further descriptions, the structure of the IL-31 protein will bedescribed regarding these four helices on canine IL-31 (SEQ ID NO: 155;Canine_IL-31), the corresponding nucleotide sequence for which is (SEQID NO: 156; Canine_IL-31) with respect to the corresponding positions onhomologous IL-31 proteins from other species (FIG. 1). Helix A iscomposed of the sequence from about amino acid 33 to 59, helix B iscomposed of the sequence from about amino acid 83 to 98, helix C iscomposed of the sequence from about amino acid 101 to 114, and helix Dis composed of the sequence from about amino acid 129 to 156. A definedloop region exists between about amino acid 97 to 101. A loop followinghelix A exists from about amino acid 57 to 62 and a loop preceding helixD from about amino acid 126 to 129. Any intervening sequence lackingpredicted secondary structure will be referred to as random coil.Treatment group 4 (ZTS-766) is a mimotope representing helices B and Cof canine IL-31 and includes an N-terminal cysteine residue tofacilitate coupling the CRM-197 carrier protein. The peptide sequencealignment of this region of the IL-31 protein is shown FIG. 16 Bcomparing canine, feline, equine, and human proteins with thecorresponding sequence reference number and amino acid positionannotated.

Titers were determined using an indirect ELISA where a full-length IL-31protein was used as the capture material for each respective assay.Serum was assayed from each study group for binding to canine and humanIL-31 proteins (FIGS. 17A and 17B respectively). Dogs vaccinated withCRM-197 conjugated full length canine IL-31 protein (T01) showed amodest increase in titer at day 42 following the second dose on day 28.This group showed a diminishing titer response for the duration of thestudy even after a third dose at day 56. Given the general response toall epitopes on the IL-31 protein (both neutralizing andnon-neutralizing), coupled with the poor titers, it is unlikely thatvaccination with a full-length IL-31 protein represents a viablecandidate for vaccine development. Group 2 from this study (ZTS-420) isa canine 15H05 mimotope with like ZTS-561 described in section 2.4however ZTS-420 is constrained by a disulphide bond between cysteinesadded at the N and C terminus of the peptide in contrast to the mT2alinker on ZTS-561. This mimotope failed to produce a robust immuneresponse when compared to the mT2a constrained form (compare FIG. 17A to15C). A modest increase in titer is observed on day 84 following thethird dose on day 56. It is possible the disulphide cyclization isinadequate or modified during CRM-197 conjugation resulting in suboptimal presentation of the immunogen to the immune cells in dogs. Group4 (ZTS-766) representing the helices B and C of canine IL-31 producedthe most robust response with titers appearing following the second doseon day 28 and increasing out to day 84 at the completion of the study.Given the IL-31 neutralizing capacity of the antibodies recognizing thissequence previously described, this mimotope represents a promisingvaccine candidate for prevention of IL-31 mediated disorders. Treatmentgroup 3 (ZTS-421) is the 15H05 epitope using the human IL-31 sequence inthis region of the mimotope. Interestingly, none of the dogs vaccinatedwith this mimotope generated a response against the canine IL-31 protein(data not shown) however an immune response was observed to the humanIL-31 protein following the second and third doses (FIG. 17B). This isremarkable specificity of the dog anti human IL-31 response given thesimilarity in sequence between the core epitope region of the 15H05mimotope (FIG. 12).

2.9. Serum Titers to IL-31 Following Immunization of Laboratory Catswith IL-31 Feline and Equine Mimotopes and Full Length Feline IL-31Protein

A serology study was performed using laboratory cats like the studydesign described herein in section 2.3 however in this study feline andequine mimotopes were compared. Laboratory cats were subcutaneouslyadministered the conjugated IL-31 mimotopes adjuvanted with a mixtureincluding the glycolipid adjuvant Bay R1005(N-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamidehydroacetate)as well as CpG oligonucleotides. A control group was included containingCRM-197 conjugated feline IL-31 (SEQ ID NO: 157; Feline_IL31_wildtype),the corresponding nucleotide sequence for which is (SEQ ID NO: 158;Feline_IL-31_wildtype) in an adjuvant mixture including the glycolipidadjuvant Bay R1005(N-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamidehydroacetate)as well as CpG oligonucleotides. (T01). 10 μg/dose of each adjuvantedmimotope or control was administered subcutaneously on days 0, 28, and56 (0.5 ml of a 20 μg/ml solution). Blood for serum was taken on day−14, 0 (pre-dose), 28 (pre-dose), 42, 56 (pre-dose), 70, and 84. Inaddition, on days 35 and 63, approximately 40 mls of blood was collectedfrom each animal into lithium heparin tubes and processed for PBMCisolation using a standard method. PBMCs were cryopreserved followingisolation until further evaluation of antigen-specific B-cells. Thestudy treatment groups are outlined in FIG. 18A. ZTS-563 (T02) isdescribed herein in section 2.4 as an immunogen that was used in theprevious dog serology study. ZTS-563 is an mT2a constrained 15H05mimotope conjugated to CRM-197. T03 (ZTS-418) is a 15H05 mimotope withequine sequence (compare the homologous canine version ZTS-420 in FIG.16A). Treatment group 4 is ZTS-423, a mimotope peptide representing theBC helix described in section 2.8 with feline IL-31 sequence. Treatmentgroup 5 is ZTS-422, a feline 15H05 mimotope with an aminohexanoic acid(Ahx) mT2b linker. FIG. 18B shows the results for the serum antibodyresponse of treatment groups T01, T02, T04 and T05 (T03 had no CRAR tofeline IL-31 protein, data not shown) on days −14, 42, and 84 to fulllength feline IL-31 using an indirect ELISA. Once again, the conjugatedform of the full-length IL-31 protein (T01) showed the poorest antibodyresponse in cats with titers to full length IL-31 never exceeding1:20000 for the duration of the study. T02 (ZTS-563) had a modestresponse throughout the study with a dose-dependent increase out to day84 indicating this presentation of the feline 15H05 epitope may be asuitable vaccine. The average titer in cats to full length feline IL-31protein following three doses of ZTS-423 (T04) are dose-dependentallyincreased to greater than 1:100,000 following the second and third doseindicating an outstanding immune response to a highly relevant epitoperegion. ZTS-422 (T05) representing the 15H05 mimotope with the AhX mT2blinker shows also shows a robust immune response in cats with titersexceeding 1:100000 following the second and third dose. The 15H05epitope in this form is clearly a relevant presentation of this regionof the IL-31 protein and represents a promising vaccine mimotope toneutralize in vivo IL-31 activity.

2.10. Sequence and Structural Considerations for the Appropriate Designof Mimotopes for Use as Vaccines

Described herein are several peptide representations of epitopes on theIL-31 protein (mimotopes) with unique sequences corresponding to theamino acids of their species of origin. The ultimate objective invaccine design is the portrayal of epitopes whereby the immune systemrecognizes them and generates a robust and specific response. Vaccinedesign is facilitated by, but not limited to, the addition of carrierproteins like CRM-197 and formulation with adjuvants. Described hereinare examples of epitopes that have been identified based on theproperties of the antibodies they are bound by. Key epitopes are thoseareas on the IL-31 protein which, when bound by an antibody, are notable to further engage the IL-31RA:OSMR receptor complex and thereforeare not able to elicit a pSTAT signaling response in cell culture or invivo. Blockade of IL-31 mediated receptor signaling is therefore anapproach to prevent and/or treat IL-31 mediated disorders like atopicdermatitis.

Mapping of antibody binding sites on proteins using mutationaltechniques is an effective way to identify key residues involved withantibody: antigen recognition as was previously described for IL-31 inU.S. Pat. No. 8,790,651 to Bammert, et al. Building upon this knowledgeenabled the design of canine and feline BC helix mimotopes which aredescribed herein as effective immunogens eliciting robust anti IL-31responses in dogs and cat. Another method using a GST canine IL-31fusion protein was described recently to map the anti-canine IL-31antibody M14 (WO 2018/156367 (Kindred Biosciences, Inc.). These authorssought to define a minimum epitope sequence recognized by the M14antibody comprised of the amino acids PSDX, X₂KI (SEQ ID NO 155, aminoacids 34-40) where X is any amino acid. Description of this sequencewith comparison to homologous IL-31 species can be found in FIG. 19A. Afurther description, including the flanking sequence surrounding theabove, is described FIG. 19B. Following identification of a minimumbinding fragment indicated in FIG. 19B (grey shaded box around aminoacids 34-42) the authors generated alanine substitutions at eachposition using a GST fusion of this peptide fragment. From these data,the above M14 minimum binding fragment was described. The fundamentalflaw with this approach is that the nature of the described bindingfragment is dependent upon its structure in the context of a GST fusionprotein. While not being bound to a single theory, it is believed thatthe amino acid sequence recognized by the M14 antibody is part of anordered alpha helical domain described herein as helix A. Alpha helicesin peptides and proteins exist, but are not limited to, the coordinationof hydrogen bonding patterns between the oxygen of carbonyl and nitrogenof amine backbone groups (Corey-Pauling rules, a dictionary ofchemistry, 2008). The minimum binding fragment described for the M14antibody is not believed to represent an adequate description of theepitope as no evidence is given to binding properties in the absence ofa GST scaffold. Furthermore, the composition of the sequencesurrounding, and including, the reported M14 epitope contains anabundance of nonpolar amino acids (I, L, V, P, G, A, M) (FIG. 19B). Thephysical properties of these amino acids in a peptide will result inaqueous insolubility and a disordered secondary structure in the absenceof intervening polar or charged amino acids. It is therefore conceivedherein that the minimum binding fragment for the M14 antibody describedin WO 2018/156367 (Kindred Biosciences, Inc.) is dependent uponproperties conferred by the GST fusion product and not inherent to thepeptide itself.

Several peptide presentations of IL-31 epitopes are described hereinwhose properties exist as independent peptides in the absence of afusion protein. This was exemplified in section 2.2 (FIG. 13B) showingthe binding and inhibitory properties of the 15H05 class of mimotopes inboth a conjugated and unconjugated form. In addition to secondarystructural features of peptides, primary amino acid sequence representsanother key aspect of vaccine design necessary for appropriatepresentation on the surface a T-cells. The appropriate amino acidssequences, in conjunction with a carrier protein having B and T cellepitopes, will elicit an immune response direct to key areas on theIL-31 protein. Multiple areas on the IL-31 have been described herein asbeing suitable to elicit a directed immune response in dogs and cats.The success of vaccine mimotopes depends on the factors described hereinand are ultimately determined in vivo by the effectiveness of theresponse. However, based on the !earnings from several epitope regionson IL-31 described here, it is conceived that other such epitopes mayexist which would make suitable vaccine mimotopes. Antibody 15H05recognizes a loop preceding helix D illustrated as site 2 on FIG. 6B. Itis conceived that other loops on the protein maybe represent epitopesaccessible by antibodies. As an example, the loop formed by theconvergence of helix A with the trailing random coil sequence sharessuch positional and structural attributes as the 15H05 loop. This ABloop is described in FIG. 20 with comparison of the primary amino acidsequences from multiple species. Not wishing to be limited to this as asingle example, it is believed that other such regions on the proteinmay share immunogenic

2.11. Serum Titers to Equine IL-31 Mimotopes Following Vaccination ofMice with Full Length Equine IL-31 Protein

Mice were immunized with full length equine IL-31 (SEQ ID NO: 165;Equine_IL-31), the corresponding nucleotide sequence for which is (SEQID NO: 166; Canine_IL-31) conjugated to CRM-197 like the methoddescribed in section 1.6 of this application. Biotin conjugated peptidesrepresenting three epitope regions described herein were designed foruse with a bio-layer interferometry binding assay (Octet, ForteBio).Description of these peptides are described in FIG. 21A. Each peptidecontains an N terminal biotin with a three-amino acid spacer sequence(GSG) annotated with bold underlined text in the figure. Thecorresponding amino acid sequence position number from SEQ ID NO 165 arealso indicated in the figure. The 15H05 mimotope includes two terminalcysteine residues (also highlighted in bold and underlined) tofacilitate cyclization by a disulphide bond. FIG. 21B shows the resultsfor a biolayer interferometry where the peptides described in FIG. 21Aare immobilized to streptavidin coated pins and then used to probemultiple dilutions of mouse anti equine IL-31 or control mouse serum.Control mouse serum was from a mouse vaccinated with an unrelatedprotein. The response, described here as the amplitude of the signalfollowing 120 seconds of antiserum association, is represented on the yaxis of the figure. From these data, the immune response resulting frompresentation of epitopes on the full equine IL-31 protein can beassessed. In addition, the ability of these IL-31 mimotopes to berecognized by those immune responses can be assessed through binding.These data indicate that all three mimotope peptides described (15H05,BC helix, and A helix) are recognized as relevant immunogens fromprocessing and presentation of the equine IL-31 protein in vivo. Minimalsignal was observed with binding of the control serum to each mimotopeexcept for the A helix which showed some dilution dependent signal. Likepresentation of mimotopes described herein which elicit immune responsesto full length protein, this experiment describes a reciprocalvalidation of these epitopes where the immune response is validated fromthe protein against the mimotope.

What is claimed is:
 1. A method of determining the identity and/oramount of an anti-IL-31 antibody in a sample, the method comprisingincubating a sample comprising an anti-IL-31 antibody with at least onemimotope selected from the group consisting of a feline IL-31 mimotope,a canine IL-31 mimotope, a horse IL-31 mimotope, and a human IL-31mimotope; and determining the identity and/or quantity of the anti-IL-31in the sample.
 2. The method of claim 1, wherein the canine IL-31mimotope is and/or comprises as part thereof the amino acid sequenceSVPADTFECKSF (SEQ ID NO: 186), SVPADTFERKSF (SEQ ID NO: 187),NSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKF (SEQ ID NO: 192),APTHQLPPSDVRKIILELQPLSRG (SEQ ID NO: 196), TGVPES (SEQ ID NO: 200) orvariants thereof that retain anti-IL-31 binding.
 3. The method of claim1, wherein the feline IL-31 mimotope is and/or comprises as part thereofthe amino acid sequence SMPADNFERKNF (SEQ ID NO: 188),NGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKF (SEQ ID NO: 193),APAHRLQPSDIRKIILELRPMSKG (SEQ ID NO: 197), IGLPES (SEQ ID NO: 201) orvariants thereof that retain anti-IL-31 binding.
 4. The method of claim1, wherein the equine IL-31 mimotope is and/or comprises as part thereofthe amino acid sequence SMPTDNFERKRF (SEQ ID NO: 189),NSSAILPYFKAISPSLNNDKSLYIIEQLDKLNF (SEQ ID NO: 194),GPIYQLQPKEIQAIIVELQNLS KK (SEQ ID NO: 198), KGVQKF (SEQ ID NO: 202) orvariants thereof that retain anti-IL-31 binding.
 5. The method of claim1, wherein the human IL-31 mimotope is and/or comprises as part thereofthe amino acid sequence SVPTDTHECKRF (SEQ ID NO: 190), SVPTDTHERKRF (SEQID NO: 191), HSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIF (SEQ ID NO: 195),LPVRLLRPSDDVQKIVEELQSLSKM (SEQ ID NO: 199), KGVLVS (SEQ ID NO: 203) orvariants thereof that retain anti-IL-31 binding.
 6. The method of claim1, wherein the mimotope is a capture reagent bound to a solid surface.7. The method of claim 6, wherein the sample is added to the mimotopecapture reagent; and secondary detection reagents are then added toquantify the amount of the antibody in the sample.
 8. The method ofclaim 1, wherein the mimotope binds to an anti-IL31 antibody orantigen-binding portion thereof that specifically binds to a region on amammalian IL-31 protein involved with interaction of the IL-31 proteinwith its co-receptor.
 9. The method of claim 8, wherein the binding ofsaid antibody to said region is impacted by mutations in a 15H05 epitopebinding region selected from the group consisting of: a) a regionbetween about amino acid residues 124 and 135 of a feline IL-31 sequencerepresented by SEQ ID NO: 157 (Feline_IL31_wildtype); b) a regionbetween about amino acid residues 124 and 135 of a canine IL-31 sequencerepresented by SEQ ID NO: 155 (Canine_IL31); and c) a region betweenabout amino acid residues 118 and 129 of an equine IL-31 sequencerepresented by SEQ ID NO: 165 (Equine_IL31).
 10. The method of claim 8,wherein the mimotope binds to an anti-IL-31 antibody or antigen-bindingportion thereof comprising at least one of the following combinations ofcomplementary determining region (CDR) sequences: 1) antibody 15H05:variable heavy (VH)-CDR1 of SYTIH (SEQ ID NO: 1), VH-CDR2 ofNINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 of WGFKYDGEWSFDV (SEQ ID NO:3), variable light (VL)-CDR1 of RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 ofKASNLHI (SEQ ID NO: 5), and VL-CDR3 of LQSQTYPLT (SEQ ID NO: 6); 2)antibody ZIL1: variable heavy (VH)-CDR1 of SYGMS (SEQ ID NO: 13),VH-CDR2 of HINSGGSSTYYADAVKG (SEQ ID NO:14), VH-CDR3 of VYTTLAAFWTDNFDY(SEQ ID NO: 15), variable light (VL)-CDR1 of SGSTNNIGILAAT (SEQ ID NO:16), VL-CDR2 of SDGNRPS (SEQ ID NO: 17, and VL-CDR3 of QSFDTTLDAYV (SEQID NO:18); 3) antibody ZIL8: VH-CDR1 of DYAMS (SEQ ID NO: 19), VH-CDR2of GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 of GFPGSFEH (SEQ ID NO:21), VL-CDR1 of TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 of YNSDRPS (SEQID NO: 23), VL-CDR3 of SVYDRTFNAV (SEQ ID NO: 24); 4) antibody ZIL9:VH-CDR1 of SYDMT (SEQ ID NO: 25), VH-CDR2 of DVNSGGTGTAYAVAVKG (SEQ IDNO: 26), VH-CDR3 of LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 of SGESLNEYYTQ(SEQ ID NO: 28), VL-CDR2 of RDTERPS (SEQ ID NO: 29), VL-CDR3 ofESAVDTGTLV (SEQ ID NO: 30); 5) antibody ZIL11: VH-CDR1 of TYVMN (SEQ IDNO: 31), VH-CDR2 of SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 ofSMVGPFDY (SEQ ID NO: 33), VL-CDR1 of SGESLSNYYAQ (SEQ ID NO: 34),VL-CDR2 of KDTERPS (SEQ ID NO: 35), VL-CDR3 of ESAVSSDTIV (SEQ ID NO:36); 6) antibody ZIL69: VH-CDR1 of SYAMK (SEQ ID NO: 37), VH-CDR2 ofTINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 of GNAESGCTGDHCPPY (SEQ IDNO: 39), VL-CDR1 of SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 of KDTERPS (SEQID NO: 41), VL-CDR3 of ESAVSSETNV (SEQ ID NO: 42); 7) antibody ZIL94:VH-CDR1 of TYFMS (SEQ ID NO: 43), VH-CDR2 of LISSDGSGTYYADAVKG (SEQ IDNO: 44), VH-CDR3 of FWRAFND (SEQ ID NO: 45), VL-CDR1 of GLNSGSVSTSNYPG(SEQ ID NO: 46), VL-CDR2 of DTGSRPS (SEQ ID NO: 47), VL-CDR3 ofSLYTDSDILV (SEQ ID NO: 48); 8) antibody ZIL154: VH-CDR1 of DRGMS (SEQ IDNO: 49), VH-CDR2 of YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 ofWDGSSFDY (SEQ ID NO: 51), VL-CDR1 of KASQSLLHSDGNTYLD (SEQ ID NO: 52),VL-CDR2 of KVSNRDP (SEQ ID NO: 53), VL-CDR3 of MQAIHFPLT (SEQ ID NO:54); 9) antibody ZIL159: VH-CDR1 of SYVMT (SEQ ID NO: 55), VH-CDR2 ofGINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 of GDIVATGTSY (SEQ ID NO:57), VL-CDR1 of SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 of KDTERPS (SEQ IDNO: 59), VL-CDR3 of KSAVSIDVGV (SEQ ID NO: 60); 10) antibody ZIL171:VH-CDR1 of TYVMN (SEQ ID NO: 61), VH-CDR2 of SINGGGSSPTYADAVRG (SEQ IDNO: 62), VH-CDR3 of SMVGPFDY (SEQ ID NO: 63), VL-CDR1 of SGKSLSYYYAQ(SEQ ID NO: 64), VL-CDR2 of KDTERPS (SEQ ID NO: 65), VL-CDR3 ofESAVSSDTIV (SEQ ID NO: 66); or 11) a variant of 1) to 10) that differsfrom respective parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69,ZIL94, ZIL154, ZIL159, or ZIL171 by addition, deletion, and/orsubstitution of one or more amino acid residues in at least one of VH orVL CDR1, CDR2, or CDR3.
 11. The method of claim 1, wherein the sample isfrom a vaccinated animal with an anti-IL-31 immune response.
 12. Themethod of claim 1, wherein the sample is from a mammal known to be orsuspected of having a puritic and/or allergic condition.